Synthesis of flavonoids and their effects on aldose reductase and sorbitol accumulation in streptozotocin-induced diabetic rat tissues

Article abstract of DOI:10.1211/0022357011775983

Aldose reductase, the key enzyme of the polyol pathway, and oxidative stress are known to play important roles in the complications of diabetes. A drug with potent inhibition of aldose reductase and oxidative stress, therefore, would be a most promising drug for the prevention of diabetic complications. The purpose of this study was to develop new compounds with these dual-effects through synthesis of chalcone derivatives and by examining the structure-activity relationships on the inhibition of rat lens aldose reductase as well as on antioxidant effects. A series of 35 flavonoid derivatives were synthesized by Winget's condensation, oxidation, and reduction of appropriate acetophenones with appropriate benzaldehydes. The inhibitory activity of these derivatives on rat lens aldose reductase and their antioxidant effects, measured using Cu²⁺ chelation and radical scavenging activities on 1,1-diphenyl-picrylhydrazyl in-vitro, were evaluated. Their effect on sorbitol accumulation in the red blood cells, lenses and sciatic nerves of streptozotocin-induced diabetic rats was also estimated. Among the new flavonoid derivatives synthesized, those with the 2′,4′-dihydroxyl groups in the A ring such as 2,4,2′,4′-tetrahydroxychalcone (22), 2,2′,4′-trihydroxychalcone (11), 2′,4′-dihydroxy-2,4-dimethylchalcone (21) and 3,4,2′,4′-tetrahydroxychalcone (18) were found to possess the highest rat lens aldose reductase inhibitory activity in-vitro, their IC50 values (concentration of inhibitors giving 50 % inhibition of enzyme activity) being 1.6 × 10^-7, 3.8 × 10^-7, 4.0 × 10^-7 and 4.6 × 10^-7 M, respectively. All of the chalcones tested except 3, 18, 23 with o-dihydroxy or hydroquinone moiety showed a weak free radical scavenging activity. In the in-vivo experiments, however, compound 18 with o-dihydroxy moiety in the B ring showed the strongest inhibitory activity in the accumulation of sorbitol in the tissues. It also showed the strongest activity in transition metal chelation and free radical scavenging activity. Of the 35 4,2′-dihydroxyl and 2′,4′-dihydroxyl derivatives of flavonoid synthesized, including chalcone, flavone, flavanone, flavonol and dihydrochalcone, some chalcone derivatives synthesized were found to possess aldose reductase inhibition and antioxidant activities in-vitro as well as inhibition in the accumulation of sorbitol in the tissues in-vivo. 3,4,2′,4′-Tetrahydroxychalcone (18, butein) was the most promising compound for the prevention or treatment of diabetic complications.

Full text of DOI:10.1211/0022357011775983

Journal of
Pharmacy and Pharmacology
JPP 2001, 53: 653–668
# 2001 The Authors
Received August 2, 2000
Accepted December 14, 2000
ISSN 0022-3573
Synthesis of flavonoids and their effects on aldose
reductase and sorbitol accumulation in streptozotocin-
induced diabetic rat tissues
Soon Sung Lim, Sang Hoon Jung, Jun Ji, Kuk Hyun Shin and
Sam Rok Keum
Abstract
Aldose reductase, the key enzyme of the polyol pathway, and oxidative stress are known to play
important roles in the complications of diabetes. A drug with potent inhibition of aldose
reductase and oxidative stress, therefore, would be a most promising drug for the prevention
of diabetic complications. The purpose of this study was to develop new compounds with these
dual-effects through synthesis of chalcone derivatives and by examining the structure–activity
relationships on the inhibition of rat lens aldose reductase as well as on antioxidant effects. A
series of 35 flavonoid derivatives were synthesized by Winget’s condensation, oxidation, and
reduction of appropriate acetophenones with appropriate benzaldehydes. The inhibitory
activity of these derivatives on rat lens aldose reductase and their antioxidant effects, measured
Natural Products Research
Institute, Seoul National
University, 28 Yeungun-dong,
Jongro-gu, Seoul, Korea
using Cu2 chelation and radical scavenging activities on 1,1-diphenyl-picrylhydrazyl in-vitro,
+
were evaluated. Their effect on sorbitol accumulation in the red blood cells, lenses and sciatic
nerves of streptozotocin-induced diabetic rats was also estimated. Among the new flavonoid
derivatives synthesized, those with the 2h,4h-dihydroxyl groups in the A ring such as 2,4,2h,4h-
tetrahydroxychalcone (22), 2,2h,4h-trihydroxychalcone (11), 2h,4h-dihydroxy-2,4-dimethylchal-
cone (21) and 3,4,2h,4h-tetrahydroxychalcone (18) were found to possess the highest rat lens
aldose reductase inhibitory activity in-vitro, their IC50 values (concentration of inhibitors giving
Soon Sung Lim, Sang Hoon Jung,
Jun Ji, Kuk Hyun Shin
Department of Chemistry, Korea
University, 126–16 Anam-dong,
Seungbuk-gu, Seoul, Korea
7
7
7
7
−
−
−
−
50% inhibition of enzyme activity) being 1.6i10 , 3.8i10 , 4.0i10 and 4.6i10 M,
respectively. All of the chalcones tested except 3, 18, 23 with o-dihydroxy or hydroquinone
moiety showed a weak free radical scavenging activity. In the in-vivo experiments, however,
compound 18 with o-dihydroxy moiety in the B ring showed the strongest inhibitory activity in
the accumulation of sorbitol in the tissues. It also showed the strongest activity in transition
metal chelation and free radical scavenging activity. Of the 35 4,2h-dihydroxyl and 2h,4h-
dihydroxyl derivatives of flavonoid synthesized, including chalcone, flavone, flavanone,
flavonol and dihydrochalcone, some chalcone derivatives synthesized were found to possess
aldose reductase inhibition and antioxidant activities in-vitro as well as inhibition in the
accumulation of sorbitol in the tissues in-vivo. 3, 4, 2h, 4h-Tetrahydroxychalcone (18, butein) was
the most promising compound for the prevention or treatment of diabetic complications.
Sam Rok Keum
Correspondence: K. H. Shin,
Natural Products Research
Institute, Seoul National
University, (110–460) 28
Yeungun-dong, Jongro-gu,
Seoul, Korea. E-mail:
khshin!plaza.snu.ac.kr
Acknowledgements and
funding: The authors are
grateful to the ONO Co. in
Japan and Professor K. Ohuchi
at Tohoku University for
epalrestat standard material
support. This work was
supported by grant No. 981-
0716-125-2 from the Basic
Research program of the Korea
Science & Engineering
Introduction
Aldose reductase, the key enzyme of the polyol pathway, has been demonstrated to
play an important role in the aetiology of the complications of diabetes such as
neuropathy, cataract formation, nephropathy and retinopathy. It has been shown
that diabetic complications can be improved by the inhibitors of aldose reductase
Foundation and partly
supported by the Brain Korea 21
Project.
653

654
Soon Sung Lim et al
in experimental animals (Beyer-Mears & Cruz 1985) as Proton (1H) and carbon (13C) nuclear magnetic res-
well as in clinical trials (Handelsman & Turtle 1981). onance spectra were determined in dimethyl sulfoxide-
There is evidence to show that diabetes is associated d6 and acetone-d6 on a Varian Gemini 2000 spectrometer
with increased oxidative stress. However, the source of at 300 and 75 MHz, respectively. Chemical shifts were
this oxidative stress remains unclear. It has been sug- reported in value from internal tetramethylsilane.
gested that glucose autoxidation and nonenzymatic Coupling constants were expressed in Hz. Infrared (IR)
glycation, together termed glycoxidation, are the major spectra were measured by Jasco FT\IR-5000. High-
contributorstotheincreaseinfreeradicalsinthediabetic resolution mass spectra (HRMS) were taken on a Jeol
lens (Wolff & Dean 1987; Srivastata et al 1996). En- JMS-AX500WA mass spectrometer. Mass spectra (MS)
2
+
dogenous protection against glycoxidation by the gluta- were taken on a HP 5989B mass spectrometer. Cu
thione redox cycle is also compromised by the com- contents were measured on a Hitachi (Zeeman-8000)
peting reduced nicotinamide dinucleotide phosphate graphite furnace atomic absorption spectrophotometer
(NADPH) requirement of elevated polyol pathway flux. (GF-AAS). Biological activities were measured on a
Aldose reductase reduction of glucose to sorbitol prob- Hitachi U-3210 spectrophotometer and Hitachi F-2000
ably contributes to oxidative stress by depleting its fluorescence spectrophotometer. Thin-layer chromato-
cofactor NADPH, which is also required for the re- graphic separations were performed with silica gel 60
generation of reduced glutathione (Lee & Chung 1999). F254 plates (Merck Art. 5715) and silica gel 60 (Merck
Aldose reductase inhibitors possessing antioxidant ac- Art. 7734; 70–230 mesh), respectively. Visualization was
tivity would therefore seem to be desirable, because it accomplished with UV light.
has been suggested that increased oxidant production
by metal-catalysed glucose oxidation may be an im-
Materials
portant mechanism in activation of aldose reductase
(Ou et al 1996). In addition, several recent reports Epalrestat
((E)-3-carboxymethyl-5-[(2E)-methyl-3-
demonstrate induction of a large number of genes phenyl-propenylidene rhodanine]) was a generous gift
including aldose reductase on exposure of cells to oxi- from Ono Pharmaceutical Co., Ltd (Japan). Reduced
dative stress (Spycher et al 1997; Carper et al 1999).
nicotinamide dinucleotide phosphate (NADPH), sor-
Naturally occurring and synthetic flavonoids have bitol dehydrogenase, -glyceraldehyde, -sorbitol,
been shown to exhibit antioxidant activity (Larson 1988) 1,1-diphenyl-2-picrylhydrazyl (DPPH), streptozotocin,
as well as inhibitory effects on aldose reductase (Shin substituted acetophenones and benzaldehydes were pur-
et al 1994, 1995; Iwata et al 1999). Moreover, Aida et chased from Aldrich Inc. (USA). Unless otherwise
al (1990) reported that isoliquiritigenin (4,2h,4h-tri- noted, all commercially available materials were used
hydroxychalcone) inhibited rat lens aldose reductase. without further purification.
Iwata et al (1999) reported that 2,4,2h,4h-tetrahydroxy-
chalcone and 2,2h,4h-trihydroxychalcone showed potent
inhibitory activity of human aldose reductase with IC50
Preparation of rat lens aldose reductase
values (concentration of inhibitors giving 50% inhi- Crude rat lens aldose reductase was prepared as follows:
bition of enzyme activity) of 7.4i10 and 1.6i10 7 , lenses were removed from Sprague-Dawley rats (250 "
9
−
−
respectively.
280 g) and frozen until use. The supernatant fraction of
In this study, we have evaluated new compounds with the rat lens homogenate was prepared according to
dual-effects through synthesis of flavonoid derivatives Hayman & Kinoshita (1965) and then partially purified
and by examining their structure–activity relationships according to Inagaki et al (1982). Partially purified
1
−
on the inhibition of rat lens aldose reductase as well enzyme with a specific activity of 6.5 mU mg was
as on antioxidant effects. The effects of the synthetic routinely used to test enzyme inhibition. The partially
derivatives on sorbitol accumulation in the tissues of purified material was separated into 1.0-mL portions
diabetic rats have been investigated also.
and stored at k40mC.
Measurements of rat lens aldose reductase
activity
Materials and Methods
General
Ratlensaldosereductaseactivitieswereassayed spectro-
Melting points (mp) were measured by a Mitamura- photometrically by measuring the decrease in absorp-
Riken melting point apparatus and were uncorrected. tion of NADPH at 340 nm over a 4-min period with

655
Synthesis of flavonoids and their effects on diabetic complications
Cu2M partitioning into n-octanol
-glyceraldehyde as a substrate (Sato & Kador 1990).
Each 1.0-mL cuvette contained equal units of enzyme,
0.10  sodium phosphate buffer (pH 6.2), 0.3 m
NADPH with or without 10 m substrate and inhibitor.
The concentration of inhibitors giving 50% inhibition
of enzyme activity (IC50) was calculated from the least-
squares regression line of the logarithmic concentrations
plotted against the remaining activity.
Stock solutions of compounds (all at 2.5 m) were
preparedin PBS in the presenceof 1 mCuSO4. Samples
(1 mL) of the compounds (vehicle alone, EDTA and
samples) were mixed with n-octanol (2 mL) and were
2
+
shaken for 10 min. After centrifugation at 1000 g, Cu
content in the organic layer was analysed by atomic
absorption spectrophotometer (Ou et al 1996).
Statistical analysis
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-
scavenging effect
The data are shown as the meanps.e.m. Significant
difference was calculated by Student’s t-test and linear
regression was analysed by the least-squares method.
The DPPH radical-scavenging effect was determined
according to the method used by Hatano et al (1989). In
microwells, 100 µL of an aqueous solution of the sample
(control:100 µL distilled water) was added to an ethanol
solution of DPPH (60 µ). Seven gradual different
concentrations (5–250 µ) were prepared for each
sample. After mixing gently and leaving to stand for
30 min at room temperature, the optical density was
determined using a microplate reader at 517 nm. The
antioxidant activity of each sample was expressed in
terms of IC50 (micromolar concentration required to
inhibit DPPH radical formation by 50%), calculated
from log-dose inhibition curves.
General procedure for the preparation of
precursors
2h,4h-Bis(methoxymethoxy)acetophenone (A)
2h,4h-Dihydroxyacetophenone (12.5 g, 82.0 mmol), an-
hydrous potassium carbonate (76 g), acetone (300 mL)
and excess chloromethylmethyl ether (7.9 mL,
102.5 mmol) were refluxed for 30 min, filtered and con-
centrated. The crude product, 2h-hydroxy-4h-methoxy-
methoxyacetophenone (a), was purified by column chro-
matography (silica gel, eluted with 20% ethyl acetate in
1
hexane) giving a pale yellow oil in 95.6% yield. H
NMR (acetone-d6, 300 MHz) δ 7.58 (dd, J l 2.1, 9.0,
1H), 6.44 (dd, J l 2.1, 9.0, 1H), 6.35 (d, J l 9.0, 1H),
6.02 (s, 1H), 3.24 (s, 1H), 2.55 (s, 1H).
In-vivo experiments (sorbitol accumulation in
streptozotocin-induced diabetic rat tissue)
A sample of a (10.0 g, 51.0 mmol) was added to a
NaH (60% in oil, 3.12 g, 130.1 mmol) suspension in
200 mL THF. This solution was stirred for 30 min in an
ice bath, and then chloromethylmethyl ether (4.8 mL,
64.0 mmol) was added. After being stirred for 30 min,
the reaction was quenched by the addition of 20 mL
distilled water. The mixture was diluted with ethyl
acetate, and then the organic phase was washed with
5% aqueous hydrochloric acid, water and brine, dried
over anhydrous magnesium sulfate, filtered and con-
centrated. The crude product was purified by column
chromatography (silica gel, eluted with 10% ethyl acet-
Diabetes was induced in male Sprague-Dawley rats
(220"250 g) by a single intraperitoneal injection of
1
streptozotocin (80 mg kg ) in sodium citrate buffer (pH
−
4.5). Control rats were injected with the vehicle only.
The animals were fed standard lab chow and water was
freely available. Two weeks after the induction of dia-
betes, the animals were administered either compounds
or vehicle alone via an intragastric tube, twice a day at
1
−
a dose of 75 mg kg \day for two weeks. Compounds
were suspended in saline containing 0.5% carmellose.
The animals were then killed under ether anaesthesia.
The contents of sorbitol in the rat red blood cells (RBC),
sciatic nerves, and lenses were determined enzymatically
(Aida et al 1988).
1
ate in hexane) giving a yellow oil in 98.5% yield. H
NMR (acetone-d6, 300 MHz) δ 7.59 (dd, J l 2.1, 9.0,
1H), 6.52 (dd, J l 2.1, 9.0, 1H), 6.47 (d, J l 9.0, 1H),
6.02 (s,1H), 5.89 (s, 1H), 3.24 (s, 1H), 3.17 (s, 1H), 2.55
(s, 1H).
Measurements of blood glucose
In each case a 200-µL blood sample was collected and 4-(Methoxymethoxy)benzaldehyde (B)
estimated for glucose by the o-toluidine method 4-Hydroxybenzaldehyde (b) (10 g, 82.0 mmol), anhy-
(Dubowski 1962).
drous potassium carbonate (76 g), acetone (300 mL)

656
Soon Sung Lim et al
and excess chloromethylmethyl ether (7.9 mL, zaldehyde (1.72 g, 9.43 mmol). After being stirred for
102.5 mmol) were refluxed for 30 min, filtered 5 h at room temperature, the reaction was quenched
and concentrated. The crude product, 4-(methoxy- with distilled water. The resulting solution was acidified
methoxy)benzaldehyde (B), was purified by column with 1  HCl, and extracted with ethyl acetate. The
chromatography (silica gel, eluted with 10% ethyl ace- organic phase was washed with brine, dried over an-
1
tate in hexane) giving a yellow oil in 98.8% yield. H hydrous magnesium sulfate and concentrated. The
NMR (acetone-d6, 300 MHz) δ 9.87 (s, 1H), 7.70 (dd, compound 10c was obtained in 91.8% isolated yield. A
J l 2.4, 8.7, 2H), 6.96 (dd, J l 2.4, 8.7, 2H), 6.02 (s, 2H), single crystallization from ethyl acetate–hexane gave the
3.24 (s, 3H).
target material as a pale yellow plate. 1H NMR
(acetone-d6, 300 MHz) δ 8.21 (d, J l 8.7, 1H), 7.97 (d, J
l 15.3, 1H), 7.87 (d, J l 15.3, 1H), 7.85 (dd, J l 2.4,
9.0, 2H), 7.4–7.5 (m, 1H) 6.64 (dd, J l 2.4, 8.7, 1H), 6.62
(dd, J l 2.1, 9.0, 2H), 6.57 (d, J l 8.7, 1H).
General procedure for the preparation of
2h-hydroxychalcone (1–24)
We synthesized 24 chalcones using Winget’s conden-
sation of appropriate acetophenones with appropriate
aromatic aldehyde (Winget et al 1969). For the synthesis
of compounds possessing a 2h,4h- or 3,4-dihydroxyl
group, the hydroxyl group of the component was pro-
tected with methoxymethyl using chloromethylmethyl
ether (Figure 1) (Rall et al 1976).
2h,4h-Dihydroxychalcone (10)
To a stirred solution of 10c (2.83 g, 8.63 mmol) in 20 mL
methanol was added 5 mL 3  HCl. After 3 h, the
reactionwasconcentratedanddilutedwithethyl acetate,
washed with water and brine, dried over magnesium
sulfate, filtered and evaporated. This crude solid was
purified by column chromatography (silica gel, eluted
with 38% ethyl acetate in hexane) to 10 (0.47 g,
2h,4h-Bis(methoxymethoxy)chalcone (10c)
1
1.97 mmol, 22.9%) as a yellow needle. H NMR (ace-
To a mixture of 2h,4h-bis(methoxymethoxy)aceto-
phenone (A) (2.0 g, 9.4 mmol) and 50% aqueous pot-
assium hydroxide solution (5.3 mL, as KOH: 2.64 g,
47.0 mmol) in 20 mL 95% ethanol was added ben-
tone-d6) δ: 13.32 (s, 1H), 8.12 (d, J l 8.7, 1H), 7.94 (d,
J l 15.3, 1H), 7.35 (d, J l 15.3, 1H), 7.44 (dd, J l 2.1,
8.7, 1H), 7.3–7.4 (m, 3H), 6.51 (dd, J l 2.1, 8.4, 1H),
6.48 (dd, J l 2.1, 8.4, 1H), 6.39 (d, J l 2.1, 1H). 13C
NMR (acetone-d6): δ 116.1 (C-1h), 159.6 (C-2h), 103.0
(C-3h), 164.5 (C-4h), 107.6 (C-5h), 132.3 (C-6h), 187.1
(CꢀO), 123.7 (C-α), 142.1 (C-β), 134.6 (C-1), 126.2 (C-
2,6), 128.4 (C-3,5), 127.7 (C-4). TLC, Rf 0.5 (5:3 hexane\
H
H
O
O
RO
CH3
HO
1
CH3 i, ii
−
i
EtOAc); IR (KBr) 3449, 1635 cm ; EI-MS (electron
+
O
OR′
O
impact mass spectrophotometry) (70 eV) m\z 240 (M ,
100), 163 (74), 137 (47), 103 (28), 77 (37) (Wollen-weber
& Seigler 1982).
OH
OCH2OCH3
OR
OH
a R = CH2OCH3 R′ = H
A R, R′ = CH2OCH3
b
B
O
R = CH2OCH3
R = H
RO
viii
4,2h-Dihydroxychalcone (1)
v
O
OR
2h-Hydroxyacetophenone (5.35 g, 35.2 mmol) and
chloromethylmethyl ether (5.26 g, 68.4 mmol) were
treated as in a to give crude 2h-methoxymethoxyaceto-
phenone (1a).
OH
RO
RO
vi
iii
OR
O
O
O
A + B
iv
RO
OR
OH
O
A mixture of crude 1a (0.63 g, 3.70 mmol), B (0.61 g,
3.70 mmol), and potassium hydroxide (1.04 g,
18.5 mmol) was treated as in 10c to give 1 (0.26 g,
O
OR
OR
1
iii
vii
A + B
2.63 mmol, 22.9%) as a yellow needle. H NMR (ace-
RO
OR
RO
OR
tone-d6, 300 MHz) δ: 12.32 (s, 1H), 8.25 (dd, J l 2.4,
8.7,1H), 7.92 (d, J l 15.3, 1H), 7.84 (d, J l 15.3, 1H),
7.82 (d, J l 9.0, 2H), 7.5–7.6 (m, 1H) 7.41 (dd, J l 2.4,
8.7, 1H), 7.06 (d, J l 9.0, 2H), 6.9–7.0 (m, 1H). 13C
NMR (acetone-d6): δ 127.4 (C-1h), 164.4 (C-2h), 116.1
(C-3h), 136.0 (C-4h), 122.0 (C-5h), 132.1 (C-6h), 205.8
O
O
Figure 1 Preparation of hydroxylated flavonoids. i. CH3OCH2Cl,
K2CO3, acetone, reflux. ii. CH3OCH2Cl, THF, NaH, 0 mC. iii. KOH,
95%ethyl hydroxide. iv. SiO2, H3BO3, piperidine, DMF. v. I2, DMSO,
reflux. vi. Methyl hydroxide, 30% H2O2, NaOH. vii. ethyl hydroxide,
10% Pd\C. viii. 3  HCl, methanol, reflux.

657
Synthesis of flavonoids and their effects on diabetic complications
(CꢀO), 120.9 (C-α), 146.6 (C-β), 127.3 (C-1), 128.9 crude 4a (0.66 g, 3.11 mmol), B (0.56 g, 3.11 mmol) and
(C-2), 116.8 (C-3), 161.3 (C-4), 116.8 (C-5), 127.3 (C-6). potassium hydroxide (0.87 g, 15.6 mmol) was treated as
TLC, Rf 0.54 (5:3 hexane\EtOAc); IR (KBr) 3320, in 10c to give 4 (0.17 g, 0.67 mmol, 24.4%) as a yellow
1
1637 cm ; EI-MS (70 eV) m\z 240 (M , 22), 146 (38), needle. 1H NMR (acetone-d6) δ: 12.67 (s, 1H), 8.16 (d,
−
+
121 (100), 119 (20), 107 (34), 91 (32) (Bhartiya & Gupta J l 8.4, 1H), 7.76 (d, J l 15.3, 1H), 7.74 (d, J l 15.3,
1982).
1H), 7.72 (d, J l 8.7, 2H), 6.93 (d, J l 8.7, 2H), 6.49 (d,
J l 2.1, 1H), 6.52 (dd, J l 2.1, 8.4, 1H), 3.87 (s, 3H).
13C NMR (acetone-d6): δ 116.7 (C-1h), 160.4 (C-2h),
4,2h,4h-Trihydroxychalcone (2)
2h,4h-Dihydroxyacetophenone (5.35 g, 35.2 mmol) and 115.9 (C-3h), 169.2 (C-4h), 116.6 (C-5h), 132.7 (C-6h),
chloromethylmethyl ether (5.26 g, 68.4 mmol) were 206.2 (CꢀO), 128.9 (C-α), 139.2 (C-β), 131.1 (C-1),
treated as in a to give crude 2h,4h-bis(methoxy- 131.8 (C-2,6), 118.5 (C-3,5), 157.0 (C-4), 56.0 (OCH3).
methoxy)acetophenone (2a). A mixture of crude 2a TLC, Rf 0.51 (5:3 hexane\EtOAc); IR (KBr) 3244,
1
−
+
(4.5 g, 18.75 mmol), B (3.42 g, 18.75 mmol) and pot- 1630 cm ; EI-MS (70 eV) m\z 270 (M , 95), 177 (33),
assium hydroxide (5.26 g, 93.8 mmol) was treated as in 151 (100), 120 (36) (Severi et al 1996).
10c to give 2 (0.66 g, 2.58 mmol, 17.4%) as a pale brown
needle. 1H NMR (acetone-d6) δ: 12.87 (s, 1H), 8.09 (d,
4,2h-Dihydroxy-5h-methoxychalcone (5)
J l 8.4, 1H), 7.79 (d, J l 15.3, 1H), 7.77 (d, J l 15.3,
2h-Hydroxy-5h-methoxyacetophenone (0.28 g,
1H), 7.72 (d, J l 8.7, 2H), 6.92 (d, J l 8.7, 2H), 6.36 (d,
1.67 mmol) and chloromethylmethyl ether (0.42 g,
J l 2.1, 1H), 6.43 (dd, J l 2.1, 8.4, 1H). 13C NMR
5.40 mmol)weretreatedasinatogivecrude5h-methoxy-
(acetone-d6): δ 116.5 (C-1h), 159.7 (C-2h), 103.1 (C-3h),
2h-methoxymethoxyacetophenone (5a). A mixture of
164.1 (C-4h), 108.8 (C-5h), 132.1 (C-6h), 187.6 (CꢀO),
crude 5a (0.64 g, 3.01 mmol), B (0.50 g, 3.01 mmol) and
123.5 (C-α), 142.9 (C-β), 127.5 (C-1), 127.6 (C-2,6),
potassium hydroxide (0.85 g, 15.05 mmol) was treated
115.3 (C-3,5), 156.3 (C-4). TLC, Rf 0.4 (1:1 hexane\
as in 10c to give 5 (0.18 g, 0.71 mmol, 24.1%) as a
yellow powder. 1H NMR (acetone-d6) δ: 13.12 (s, 1H),
1
−
EtOAc); IR (KBr) 3200, 1630 cm ; EI-MS (70 eV) m\z
+
256 (M , 44), 137 (53), 120 (130), 91 (17) (Grasselli &
8.05 (d, J l 2.1, 1H), 7.78 (d, J l 15.3, 1H), 6.94 (d, J l
15.3, 1H), 7.75 (d, J l 8.7, 2H), 7.36 (d, J l 8.7, 2H),
Ritchey 1975).
6.85 (d, J l 8.4, 1H), 6.39 (dd, J l 2.1, 8.4, 1H), 3.92 (s,
4,2h,5h-Trihydroxychalcone (3)
3H). 13C NMR (acetone-d6): δ 124.9 (C-1h), 150.8 (C-2h),
2h,5h-Dihydroxyacetophenone (0.42 g, 2.75 mmol) and
117.2 (C-3h), 121.3 (C-4h), 155.2 (C-5h), 116.7 (C-6h),
chloromethylmethyl ether (0.72 g, 9.3 mmol) were
187.0 (CꢀO), 123.3 (C-α), 142.8 (C-β), 127.5 (C-1),
treated as in a to give crude 2h,5h-bis(methoxy-
127.6 (C-2,6), 115.6 (C-3,5), 156.5 (C-4), 54.7 (OCH3).
methoxy)acetophenone (3a). A mixture of crude 3a
TLC, Rf 0.59 (5:3 hexane\EtOAc); IR (KBr) 3254,
(0.5 g, 2.08 mmol), B (0.38 g, 2.08 mmol) and potassium
1
−
+
1637 cm ; EI-MS (70 eV) m\z 270 (M , 24), 167 (31),
149 (83), 79 (79), 57 (100); HR-MS (EI) exact mass for
hydroxide (0.58 g, 10.4 mmol) was treated as in 10c to
give 3 (0.15 g, 0.59 mmol, 21.1%) as a brown needle.
1H NMR (acetone-d6) δ: 12.87 (s, 1H), 8.11 (d, J l 2.1,
1H), 7.87 (d, J l 15.3, 1H), 7.56 (d, J l 15.3, 1H), 7.58
+
C16H14O4 (M ) 270.0892, found 270.0868.
(d, J l 8.7, 2H), 7.09 (d, J l 8.7, 2H), 6.91 (dd, J l 2.1, 4,2h-Dihydroxy-6h-methoxychalcone (6)
8.4, 1H), 6.81 (d, J l 8.4, 1H). 13C NMR (acetone-d6): 2h-Hydroxy-6h-methoxyacetophenone (0.31 g,
δ 125.3 (C-1h), 146.4 (C-2h), 116.7 (C-3h), 132.0 (C-4h), 1.83 mmol) and chloromethylmethyl ether (0.42 g,
119.4 (C-5h), 132.1 (C-6h), 206.2 (CꢀO), 119.4 (C-α), 5.40 mmol) were treated as in a to give crude 2h-meth-
132.1 (C-β), 125.4 (C-1), 125.5 (C-2,6), 115.5 (C-3,5), oxymethoxy-6h-methoxyacetophenone (6a). A mixture
146.4 (C-4). TLC, Rf 0.46 (1:1 hexane\EtOAc); IR of crude 6a (0.66 g, 1.85 mmol), B (0.56 g, 3.11 mmol)
1
−
+
(KBr) 3364, 1610 cm ; EI-MS (70 eV) m\z 256 (M , and potassium hydroxide (0.87 g, 15.6 mmol) was
61), 137 (100), 120 (63), 105 (23), 91 (63) (Bhartiya & treated as in 10c to give 6 (0.14 g, 0.55 mmol, 28.9%) as
Gupta 1982).
a brown needle. 1H NMR (acetone-d6) δ: 12.62 (s, 1H),
7.86 (d, J l 15.1, 1H), 7.74 (d, J l 15.1, 1H), 7.53 (d, J
l 8.7, 2H), 6.98 (d, J l 8.7, 2H), 6.63 (brd, J l 8.4,
4,2h-Dihydroxy-4h-methoxychalcone (4)
2h-Hydroxy-4h-methoxyacetophenone (0.43 g, 1H), 7.29 (t, J l 8.4, 1H), 6.41 (brd, J l 8.4, 1H), 3.97(s,
2.61 mmol) and chloromethylmethyl ether (0.42 g, 3H). 13C NMR (acetone-d6): δ 116.8 (C-1h), 147.7 (C-
5.39 mmol)weretreatedasinatogivecrude2h-methoxy- 2h), 117.6 (C-3h), 133.1 (C-4h), 116.7 (C-5h), 164.6 (C-6h),
methoxy-4h-methoxyacetophenone (4a). A mixture of 205.9 (CꢀO), 147.6 (C-α), 117.5 (C-β), 121.1 (C-1),

658
Soon Sung Lim et al
121.2 (C-2,6), 116.7 (C-3,5), 139.5 (C-4), 57.1 (OCH3). treated as in a to give crude 5h-bromo-2h-methoxy-
TLC, Rf 0.35 (5:3 hexane\EtOAc); IR (KBr) 3331, methoxyacetophenone (9a). A mixture of crude 9a
1
−
+
1666 cm ; EI-MS (70 eV) m\z 270 (M , 100), 177 (38), (0.64 g, 3.30 mmol), B (0.71 g, 3.30 mmol) and pot-
151 (98), 120 (32) (Severi et al 1996).
assium hydroxide (0.92 g, 16.5 mmol) was treated as in
10c to give 9 (0.18 g, 0.55 mmol, 22.5%) as a yellow
1
plate. H NMR (acetone-d6) δ: 12.56 (s, 1H), 8.38 (d,
4,2h-Dihydroxy-5h-methylchalcone (7)
J l 2.4, 1H), 7.94 (d, J l 15.3, 1H), 7.81 (d, J l 15.3,
1H), 7.66 (d, J l 8.7, 2H), 7.01 (d, J l 8.7, 2H), 6.92 (d,
J l 8.7, 1H), 6.95 (dd, J l 2.4, 8.7, 1H). 13C NMR
(acetone-d6): δ 126.1 (C-1h), 157.5 (C-2h), 118.4 (C-3h),
139.0 (C-4h), 116.2 (C-5h), 134.4 (C-6h), 187.7 (CꢀO),
123.2 (C-α), 142.4 (C-β), 127.7 (C-1), 127.6 (C-2,6),
115.4 (C-3,5), 156.5 (C-4). TLC, Rf 0.46 (5:3 hexane\
2h-Hydroxy-5h-methylacetophenone (0.34 g, 2.28 mmol)
and chloromethylmethyl ether (0.54 g, 7.02 mmol) were
treated as in a to give crude 2h-methoxymethoxy-5h-
methylacetophenone (7a). A mixture of crude 7a (0.64 g,
3.30 mmol), B (0.61 g, 3.3 mmol) and potassium hy-
droxide (0.92 g, 16.5 mmol) was treated as in 10c to give
1
7 (0.12 g, 0.45 mmol, 19.8%) as a pale red powder. H
1
−
EtOAc); IR (KBr) 3427, 1637 cm ; EI-MS (70 eV) m\z
NMR (acetone-d6) δ: 13.23 (s, 1H), 8.13 (s, 1H), 7.88 (d,
J l 15.3, 1H), 7.76 (d, J l 15.3, 1H), 7.38 (d, J l 8.7,
2H), 7.13 (d, J l 8.7, 2H), 6.87 (d, J l 8.4, 1H), 6.93 (d,
J l 8.4, 1H), 2.86 (s, 3H). 13C NMR (acetone-d6): δ
123.8 (C-1h), 155.5 (C-2h), 116.1 (C-3h), 136.4 (C-4h),
130.8 (C-5h), 131.8 (C-6h), 187.0 (CꢀO), 123.9 (C-α),
142.8 (C-β), 127.4 (C-1), 127.6 (C-2,6), 115.1 (C-3,5),
156.5 (C-4), 20.3 (CH3). TLC, Rf 0.56 (5:3 hexane\
+
318 (M , 47), 201 (25), 147 (10), 120 (100), 91 (31); HR-
+
MS (EI) exact mass for C15H11O3Br (M ) 317.9892,
found 317.9868.
2,2h,4h-Trihydroxychalcone (11)
2-Hydroxybenzaldehyde (0.99 g, 8.09 mmol) and chlo-
romethylmethyl ether (1.3 g, 17.1 mmol) were treated as
in b to give crude 2-methoxymethoxybenzaldehyde
(11b).
1
−
EtOAc); IR (KBr) 3350, 1637 cm ; EI-MS (70 eV)
+
m\z 254 (M , 1.3), 150 (15), 135 (100), 107 (12), 77 (15);
+
HR-MS (EI) exact mass for C16H14O3 (M ) 254.0943,
A mixture of A (2.0 g, 9.4 mmol), crude 11b (1.72 g,
9.4 mmol) and potassium hydroxide (2.64 g, 47.0 mmol)
was treated as in 10c to give 11 (0.35 g, 1.38 mmol,
17.0%) as a red needle. 1H NMR (acetone-d6) δ: 13.12
(s, 1H), 8.04 (d, J l 8.7, 1H), 8.25 (d, J l 15.1, 1H), 7.95
(d, J l 15.1, 1H), 6.95 (d, J l 2.1, 1H), 7.54 (dd, J l 2.1,
8.7, 1H), 7.2–7.3 (m, 1H), 7.74 (d, J l 8.7, 1H), 6.37 (d,
J l 8.7, 1H), 6.47 (d, J l 2.1, 1H). 13C NMR (acetone-
d6): δ 116.2 (C-1h), 164.4 (C-2h), 103.6 (C-3h), 167.4 (C-
4h), 108.7 (C-5h), 132.6 (C-6h), 192.9 (CꢀO), 129.8 (C-α),
140.9 (C-β), 122.6 (C-1), 157.8 (C-2), 116.9 (C-3), 129.8
(C-4), 120.7 (C-5), 127.4 (C-6). TLC, Rf 0.34 (5:3
found 254.0962.
5h-Chloro-4,2h-dihydroxychalcone (8)
2h-Hydroxy-5h-chloroacetophenone (2.0 g, 2.69 mmol)
and chloromethylmethyl ether (0.54 g, 7.02 mmol) were
treated as in a to give crude 5h-chloro-2h-methoxy-
methoxyacetophenone (8a). A mixture of crude 8a
(0.64 g, 3.30 mmol), B (0.61 g, 3.30 mmol) and pot-
assium hydroxide (0.92 g, 16.5 mmol) was treated as in
10c to give 8 (0.13 g, 0.46 mmol, 17.3%) as a yellow
needle. 1H NMR (acetone-d6) δ: 13.15 (s, 1H), 8.24 (d,
J l 2.1, 1H), 7.93 (d, J l 15.3, 1H), 7.88 (d, J l 15.3,
1H), 7.79 (d, J l 8.7, 2H), 7.50 (d, J l 8.7, 2H), 6.98 (d,
J l 8.4, 1H), 6.92 (dd, J l 2.1, 8.4, 1H). 13C NMR
(acetone-d6): δ 125.1 (C-1h), 162.9 (C-2h), 118.2 (C-3h),
132.7 (C-4h), 127.9 (C-5h), 131.0 (C-6h), 190.9 (CꢀO),
125.0 (C-α), 146.6 (C-β), 127.3 (C-1), 127.6 (C-2,6),
115.4 (C-3,5), 161.2 (C-4). TLC, Rf 0.43 (5:3 hexane\
1
−
hexane\EtOAc); IR (KBr) 3393, 1630 cm ; EI-MS
+
(70 eV) m\z 256 (M , 49), 147 (10), 137 (100), 118 (19),
91 (83) (Geissman & Clinton 1946).
2h,4h-Dihydroxy-4-methoxychalcone (12)
A mixture of A (3.60 g, 16.94 mmol), 4-methoxy-
benzaldehyde (2.27 g, 16.94 mmol) and potassium hy-
droxide (5.84 g, 104.0 mmol) was treated as in 10c to
give 12 (1.10 g, 4.08 mmol, 24.1%) as a yellow needle.
1H NMR (acetone-d6) δ: 13.22 (s, 1H), 8.12 (d, J l 8.7,
1H), 7.80 (d, J l 15.3, 1H), 7.70 (d, J l 15.3, 1H), 7.49
(d, J l 8.7, 2H), 7.01 (d, J l 8.7, 2H), 6.36 (d, J l 2.1,
1H), 6.46 (dd, J l 2.1, 8.7, 1H), 3.84 (s, 3H). 13C NMR
1
−
EtOAc); IR (KBr) 3368, 1637 cm ; EI-MS (70 eV) m\z
+
274 (M , 35), 155 (27), 120 (100), 107 (16); HR-MS (EI)
+
exact mass for C15H11O3Cl (M ) 274.0397, found
274.0372.
5h-Bromo-4,2h-dihydroxychalcone (9)
5h-Bromo-2h-hydroxyacetophenone (0.53 g, 2.45 mmol) (acetone-d6): δ 115.7 (C-1h), 159.3 (C-2h), 103.2 (C-3h),
and chloromethylmethyl ether (0.42 g, 5.39 mmol) were 164.3 (C-4h), 108.1 (C-5h), 132.2 (C-6h), 187.3 (CꢀO),

659
Synthesis of flavonoids and their effects on diabetic complications
123.4 (C-α), 142.7 (C-β), 127.2 (C-1), 127.3 (C-2,6), (acetone-d6): δ 116.2 (C-1h), 159.3 (C-2h), 103.3 (C-3h),
114.0 (C-3,5), 161.2 (C-4), 56.8 (OCH3). TLC, Rf 0.55 163.5 (C-4h), 108.5 (C-5h), 132.6 (C-6h), 189.0 (CꢀO),
1
−
(5:3 hexane\EtOAc); IR (KBr) 3422, 1635 cm ; EI- 123.4 (C-α), 141.8 (C-β), 131.9 (C-1), 126.1 (C-2,6),
+
MS (70 eV) m\z 270 (M , 67), 163 (12), 134 (77), 121 129.1 (C-3,5), 136.9 (C-4), 19.8 (CH3). TLC, Rf 0.59 (5:3
1
+
−
(100); HR-MS (EI) exact mass for C16H14O4 (M ) hexane\EtOAc); IR (KBr) 3267, 1631 cm ; EI-MS
+
270.0892, found 270.0881.
(70 eV) m\z 254 (M , 100), 163 (61), 137 (47), 118 (45),
+
91 (22); HR-MS (EI) exact mass for C16H14O3 (M )
254.0943, found 254.0976.
4-Bromo-2h,4h-dihydroxychalcone (13)
A mixture of A (5.0 g, 20.8 mmol), 4-bromobenzalde-
hyde (2.58 mL, 20.8 mmol) and potassium hydroxide 2h,4h-Dihydroxy-4-dimethylaminochalcone (16)
(5.84 g, 104.0 mmol) was treated as in 10c to give 13 A mixture of A (1.0 g, 4.16 mmol), 4-dimethylamino-
(2.67 g, 8.36 mmol, 35.8%) as a yellow plate. 1H NMR benzaldehyde (0.62 g, 4.16 mmol) and potassium hy-
(acetone-d6) δ: 13.57 (s, 1H), 8.14 (d, J l 8.7, 1H), 7.98 droxide (1.17 g, 20.8 mmol) was treated as in 10c to give
(d, J l 15.3, 1H), 7.74 (d, J l 15.3, 1H), 7.81 (d, J l 8.7, 16 (0.24 g, 0.86 mmol, 25.24%) as a dark brown needle.
2H), 7.64 (d, J l 8.7, 2H), 6.38 (d, J l 2.1, 1H), 6.47 1H NMR (acetone-d6) δ: 12.42 (s, 1H), 7.86 (d, J l 8.7,
(dd, J l 2.1, 8.7, 1H). 13C NMR (acetone-d6): δ 116.4 1H), 7.82 (d, J l 15.3, 1H), 7.67 (d, J l 15.3, 1H), 7.34
(C-1h), 159.1 (C-2h), 103.1 (C-3h), 164.2 (C-4h), 108.0 (C- (d, J l 8.7, 2H), 6.39 (d, J l 8.7, 2H), 6.32 (d, J l 2.1,
5h), 132.4 (C-6h), 187.4 (CꢀO), 123.6 (C-α), 142.3 (C-β), 1H), 6.37 (dd, J l 2.1, 8.7, 1H), 3.04 (s, 3H), 3.03(s, 3H).
133.9 (C-1), 128.4 (C-2,6), 131.7 (C-3,5), 122.3 (C-4). 13C NMR (acetone-d6): δ 116.7 (C-1h), 159.5 (C-2h),
TLC, Rf 0.5 (1:1 hexane\EtOAc); IR (KBr) 3447, 103.6 (C-3h), 165.4 (C-4h), 107.9 (C-5h), 132.9 (C-6h),
1
−
+
1637 cm ; EI-MS (70 eV) m\z 317 (M , 14), 181 (37), 187.9 (CꢀO), 123.8 (C-α), 142.0 (C-β), 124.4 (C-1),
137 (29), 102 (100); HR-MS (EI) exact mass for 127.1 (C-2,6), 113.0 (C-3,5), 143.7 (C-4). TLC, Rf 0.5
1
+
C15H11O3Br (M ) 317.9892, found 317.9866.
−
(5:3 hexane\EtOAc); IR (KBr) 3294, 1660 cm ; EI-
+
MS (70 eV) m\z 283 (M , 49), 147 (100), 134 (84), 77
+
(15); HR-MS (EI) exact mass for C17H17O3N (M )
2h,4h-Dihydroxy-4-nitrochalcone (14)
A mixture of A (2.5 g, 9.18 mmol), 4-nitrobenzaldehyde
(1.38 g, 9.18 mmol) and potassium hydroxide (2.58 g,
283.1208, found 283.1263.
45.9 mmol) was treated as in 10c to give 14 (0.14 g, 2h,4h-Dihydroxy-3,4-dimethoxychalcone (17)
0.49 mmol, 8.84%) as a brown powder. 1H NMR A mixture of A (5.0 g, 20.8 mmol), 3,4-dimethoxy-
(acetone-d6) δ 13.78 (s, 1H), 8.32 (d, J l 8.7, 1H), 8.18 benzaldehyde (3.46 g, 20.8 mmol) and potassium hy-
(d, J l 15.3, 1H), 7.95 (d, J l 15.3, 1H), 8.14 (d, J l 8.7, droxide (5.84 g, 104.0 mmol) was treated as in 10c to
2H), 8.29 (d, J l 8.7, 2H), 6.60 (s, 1H), 6.64 (d, J l 8.7, give 17 (0.52 g, 1.73 mmol, 11.3%) as a yellow plate.
1H). 13C NMR (acetone-d6): δ 115.2 (C-1h), 164.4 (C- 1H NMR (acetone-d6) δ: 12.98 (s, 1H), 8.08 (d, J l 8.1,
2h), 103.8 (C-3h), 165.5 (C-4h), 108.9 (C-5h), 134.6 (C-6h), 1H), 7.88 (d, J l 15.1, 1H), 7.68 (d, J l 15.1, 1H), 7.52
203.9 (CꢀO), 124.1 (C-α), 133.6 (C-β), 147.7 (C-1), (d, J l 8.1, 1H), 7.01 (dd, J l 2.1, 8.1, 1H), 7.26 (d, J l
127.6 (C-2,6), 123.9 (C-3,5), 152.9 (C-4). TLC, Rf 0.48 2.1, 1H), 6.36 (d, J l 2.1, 1H), 6.44 (dd, J l 2.1, 8.4,
(5:3 hexane\EtOAc); IR (KBr) 3395, 1628 cm ; EI- 1H), 3.87 (s, 3H), 3.85 (s, 3H). 13C NMR (acetone-d6):
1
−
+
MS (70 eV) m\z 285 (M , 17), 163 (20), 137 (100), 77 δ 116.9 (C-1h), 159.2 (C-2h), 103.2 (C-3h), 164.3 (C-4h),
(33) (Jorge & Sbarbati-Nudelman 1986).
108.6 (C-5h), 132.2 (C-6h), 187.2 (CꢀO), 122.9 (C-α),
141.9 (C-β), 128.2 (C-1), 112.8 (C-2), 147.5 (C-3), 146.8
(C-4), 115.0 (C-5), 119.5 (C-6), 54.5 (OCH3), 54.2
(OCH3). TLC, Rf 0.56 (5:3 hexane\EtOAc); IR (KBr)
2h,4h-Dihydroxy-4-methylchalcone (15)
A mixture of A (1.0 g, 4.16 mmol), 4-methylbenzal- 3423, 1637 cm ; EI-MS (70 eV) m\z 300 (M , 73), 191
dehyde (0.50 g, 4.16 mmol) and potassium hydroxide (100), 137 (73), 102 (81); HR-MS (EI) exact mass for
–1
+
+
(1.17 g, 20.8 mmol) was treated as in 10c to give 15 C17H16O5 (M ) 300.3024, found 300.3052.
1
(0.10 g, 0.39 mmol, 16.0%) as a yellow powder. H
NMR (acetone-d6) δ: 13.08 (s, 1H), 8.12 (d, J l 8.7,
1H), 7.88 (d, J l 15.3, 1H), 7.67 (d, J l 15.3, 1H), 7.85 3,4,2h,4h-Tetrahydroxychalcone (18)
(d, J l 8.7, 2H), 7.26 (d, J l 8.7, 2H), 6.37 (d, J l 2.1, 3, 4-Dihydroxybenzaldehyde (1.11 g, 8.16 mmol) and
1H), 6.47 (dd, J l 2.1, 8.4, 1H), 2.35 (s, 3H). 13C NMR chloromethylmethyl ether (1.82 g, 23.9 mmol) were

660
Soon Sung Lim et al
treated as in b to give crude 3, 4-bis(methoxy- 89), 179 (91), 151 (100), 137 (75); HR-MS (EI) exact
+
methoxy)benzaldehyde (18b). A mixture of A (2.5 g, mass for C16H13O4F (M ) 288.0798, found 288.0785.
9.18 mmol), crude 18b (2.0 g, 9.18 mmol) and potassium
hydroxide (2.58 g, 45.9 mmol) was treated as in 10c to
give 18 (0.54 g, 1.97 mmol, 21.5%) as a pale red needle.
2h,4h-Dihydroxy-2,4-dimethylchalcone (21)
A mixture of A (1.0 g, 4.17 mmol), 2,4-dimethyl-
1H NMR (acetone-d6) δ: 13.75 (s, 1H), 8.10 (d, J l 8.4,
benzaldehyde (0.56 g, 4.17 mmol) and potassium hy-
1H), 7.74 (d, J l 15.3, 1H), 7.50 (d, J l 15.3, 1H), 7.17
droxide (1.27 g, 20.8 mmol) was treated as in 10c to give
(dd, J l 2.1, 8.4, 1H), 6.81 (d, J l 2.1, 1H), 7.12 (dd, J
1
21 (0.24 g, 0.91 mmol, 29.4%) as a brown needle. H
l 2.1, 8.4, 1H), 6.35 (d, J l 8.4, 1H), 6.45 (d, J l 2.1,
NMR (acetone-d6) δ: 12.89 (s, 1H), 7.47 (d, J l 8.4,
1H). 13C NMR (acetone-d6): δ 116.1 (C-1h), 163.3 (C-
1H), 8.17 (d, J l 15.3, 1H), 7.39 (d, J l 15.3, 1H), 6.81
2h), 110.9 (C-3h), 134.0 (C-4h), 114.4 (C-5h), 118.9 (C-6h),
(d, J l 2.1, 1H), 6.89 (dd, J l 2.1, 8.4, 1H), 7.06 (dd, J
206.4 (CꢀO), 118.1 (C-α), 146.1 (C-β), 129.2 (C-1),
114.4 (C-2), 145.3 (C-3), 146.2 (C-4), 130.2 (C-5), 126.7
l 2.1, 8.4, 1H), 6.39 (d, J l 8.4, 1H), 6.48 (d, J l 2.1,
1H), 2.43 (s, 3H), 2.46 (s, 3H). 13C NMR (acetone-d6):
(C-6). TLC, Rf 0.43 (1:1 hexane\EtOAc); IR (KBr)
δ 115.5 (C-1h), 164.4 (C-2h), 103.7 (C-3h), 166.9 (C-4h),
108.6 (C-5h), 132.6 (C-6h), 192.8 (CꢀO), 127.4 (C-α),
142.3 (C-β), 132.6 (C-1), 137.6 (C-2), 129.0 (C-3), 139.1
–1
+
3335, 1637 cm ; EI-MS (70 eV) m\z 272 (M , 100), 163
(26), 150 (38), 137 (94) (Price 1939).
(C-4), 127.4 (C-5), 126.9 (C-6), 21.3 (CH3), 19.7 (CH3).
TLC, Rf 0.52 (5:3 hexane\EtOAc); mp 110–112mC
3,4-Dichloro-2h,4h-dihydroxychalcone (19)
A mixture of A (1.0 g, 4.16 mmol), 3,4-dichlorobenz-
1
(CH3OH); IR (KBr) 3410, 1630 cm ; EI-MS (70 eV)
−
+
m\z 268 (M , 52), 250 (100), 163 (29), 137 (66), 115 (49);
aldehyde (0.74 g, 4.16 mmol) and potassium hydroxide
(1.17 g, 20.8 mmol) was treated as in 10c to give 19
(0.43 g, 1.39 mmol, 33.6%) as a yellow needle. 1H NMR
+
HR-MS (EI) exact mass for C17H16O3 (M ) 268.1099,
found 268.1065.
(acetone-d6) δ: 12.56 (s, 1H), 8.17 (d, J l 8.4, 1H), 8.05
(d, J l 15.3, 1H), 7.71 (d, J l 15.3, 1H), 8.11 (dd, J l
2,4,2h,4h-Tetrahydroxychalcone (22)
2, 4-Dihydroxybenzaldehyde (2.19 g, 15.9 mmol) and
2.1, 8.4, 1H), 7.65 (d, J l 8.4, 1H), 7.81 (d, J l 2.1, 1H),
6.38 (d, J l 2.1, 1H), 6.47 (dd, J l 2.1, 8.4, 1H). 13C
chloromethylmethyl ether (2.48 g, 34.2 mmol) were
treated as in b to give crude 2,4-bis(methoxy-
NMR (acetone-d6): δ 115.9 (C-1h), 159.0 (C-2h), 103.4
(C-3h), 163.5 (C-4h), 108.9 (C-5h), 132.1 (C-6h), 187.8
methoxy)benzaldehyde (22b). A mixture of A (4.92 g,
(CꢀO), 123.1 (C-α), 142.0 (C-β), 134.4 (C-1), 128.0 (C-
2), 134.1 (C-3), 133.4 (C-4), 130.2 (C-5), 126.7 (C-6).
20.4 mmol), crude 22b (4.6 g, 20.4 mmol) and potassium
hydroxide (5.72 g, 102.0 mmol) was treated as in 10c to
TLC, Rf 0.36 (5:3 hexane\EtOAc); IR (KBr) 3200,
give 22 (1.24 g, 4.56 mmol, 28.6%) as a pale red needle.
1H NMR (acetone-d6) δ: 12.56 (s, 1H), 8.02 (d, J l 8.4,
1H), 8.22 (d, J l 15.3, 1H), 7.78 (d, J l 15.3, 1H), 6.51
(d, J l 2.1, 1H), 7.66 (d, J l 8.4, 1H), 7.74 (dd, J l 2.1,
8.4, 1H), 6.34 (d, J l 2.1, 1H), 6.43 (dd, J l 2.1, 8.4,
1H). 13C NMR (acetone-d6): δ 116.8 (C-1h), 165.6 (C-
–1
+
1630 cm ; EI-MS (70 eV) m\z 310 (M , 46), 163 (100),
136 (78), 108 (37), 81 (24); HR-MS (EI) exact mass for
+
C16H14O3Cl (M ) 324.0320, found 324.0312.
3-Fluoro-2h,4h-dihydroxy-4-methoxychalcone (20)
A mixture of A (1.0 g, 4.16 mmol), 3-fluoro-4-methoxy-
2h), 108.8 (C-3h), 164.3 (C-4h), 110.9 (C-5h), 133.6 (C-6h),
195.2 (CꢀO), 125.0 (C-α), 149.4 (C-β), 113.9 (C-1),
benzaldehyde (0.68 g, 4.06 mmol) and potassium hy-
162.5 (C-2), 103.0 (C-3), 162.5 (C-4), 108.7 (C-5), 129.4
droxide (1.17 g, 20.8 mmol) was treated as in 10c to give
1
20 (0.48 g, 1.67 mmol, 30.6%) as a yellow needle. H
(C-6). TLC, Rf 0.35 (1:3 hexane\EtOAc); IR (KBr)
–1
+
3366, 1624 cm ; EI-MS (70 eV) m\z 272 (M , 24),
167 (31), 149 (83), 79 (79), 55 (100) (Delle-Monache et al
1995).
NMR (acetone-d6) δ: 12.53 (s, 1H), 8.15 (d, J l 8.4,
1H), 7.87 (d, J l 15.3, 1H), 7.60 (d, J l 15.3, 1H), 7.84
(s, 1H), 7.18 (dd, J l 2.1, 8.4, 1H), 7.49 (d, J l 8.4, 1H),
6.37 (d, J l 2.1, 1H), 6.46 (d, J l 8.4, 1H), 3.80 (s, 3H).
13C NMR (acetone-d6): δ 116.5 (C-1h), 159.1 (C-2h), 2,5,2h,4h-Tetrahydroxychalcone (23)
103.2 (C-3h), 164.6 (C-4h), 107.9 (C-5h), 132.5 (C-6h), 2,5-Dihydroxybenzaldehyde (0.36 g, 2.64 mmol) and
187.4 (CꢀO), 123.8 (C-α), 142.5 (C-β), 128.8 (C-1), chloromethylmethyl ether (0.46 g, 5.98 mmol) were
114.2 (C-2), 147.6 (C-3), 148.2 (C-4), 115.6 (C-5), 122.8 treated as in b to give crude 2,5-bis(methoxy-
(C-6), 57.5 (OCH3). TLC, Rf 0.55 (1:1 hexane\EtOAc); methoxy)benzaldehyde (23b). A mixture of A (1.80 g,
1
−
+
IR (KBr) 3250, 1640 cm ; EI-MS (70 eV) m\z 288 (M , 4.08 mmol), crude 23b (0.9 g, 4.08 mmol) and potassium

661
Synthesis of flavonoids and their effects on diabetic complications
hydroxide (2.85 g, 20.4 mmol) was treated as in 10c to with water and brine, dried over magnesium sulfate,
give 23 (0.17 g, 0.63 mmol, 23.9%) as a yellow needle. filtered and evaporated. This crude solid was purified by
1H NMR (acetone-d6) δ: 13.87 (s, 1H), 8.07 (d, J l 8.4, column chromatography (silica gel, eluted with 10%
1H), 8.20 (d, J l 15.3, 1H), 7.86 (d, J l 15.3, 1H), 6.82 ethylacetate inhexane) to 25(0.16 g, 0.63 mmol, 43.5%)
1
(d, J l 8.4, 1H), 7.24 (d, J l 8.4, 1H), 7.97 (s, 1H), 6.36 as a yellow needle. H NMR (acetone-d6, 300 MHz) δ:
(dd, J l 2.1, 8.4, 1H), 6.47 (dd, J l 2.1, 8.4, 1H). 13C 11.80 (s, 1H), 8.02 (s, 1H), 6.74 (d, J l 8.7, 1H), 6.65 (d,
NMR (acetone-d6): δ 116.1 (C-1h), 159.4 (C-2h), 103.6 J l 8.7, 1H), 7.33 (d, J l 8.4, 2H), 6.99 (d, J l 8.4, 2H),
(C-3h), 164.7 (C-4h), 108.6 (C-5h), 131.7 (C-6h), 187.9 3.26 (t, J l 7.3, 2H), 2.92 (t, J l 7.3, 2H). 13C NMR
(CꢀO), 123.4 (C-α), 142.9 (C-β), 123.5 (C-1), 147.6 (acetone-d6): δ 126.0 (C-1h), 150.0 (C-2h), 117.0 (C-3h),
(C-2), 117.0 (C-3), 116.3 (C-4), 149.8 (C-5), 114.8 (C-6). 121.5 (C-4h), 149.8 (C-5h), 117.2 (C-6h), 196.7 (CꢀO),
TLC, Rf 0.45 (1:3 hexane\EtOAc); IR (KBr) 3391, 41.5 (C-α), 29.9 (C-β), 132.8 (C-1), 129.3 (C-2), 115.6
1
−
+
1637 cm ; EI-MS (70 eV) m\z 272 (M , 14), 137 (100), (C-3), 154.5 (C-4), 115.6 (C-5), 129.3 (C-6). TLC, Rf
1
−
107 (20), 81 (33), 55 (53); HR-MS (EI) exact mass for 0.48 (5:3 hexane\EtOAc); IR (KBr) 3434, 1643 cm ;
+
C15H12O5 (M ) 272.0685, found 272.0639.
+
EI-MS (70 eV) m\z 256 (M , 18), 149 (42), 137 (100),
129 (26), 119 (40); HR-MS (EI) exact mass for C15H14O4
+
(M ) 258.0892, found 258.0839.
2,3,4,2h,4h-Pentahydroxychalcone (24)
2, 3, 4-Trihydroxybenzaldehyde (0.38 g, 2.44 mmol) and
chloromethylmethyl ether (0.58 g, 7.32 mmol) were 2,2h,4h-Trihydroxydihydrochalcone (26)
treated as in b to give crude 2, 3, 4-Tris(methoxy- A mixture of 2,2h,4h-Tris(methoxymethoxy)chalcone
methoxy)benzaldehyde (24b). A mixture of A (1.0 g, (0.5 g, 1.45 mmol), 10% palladium on charcoal (5 mg)
4.16 mmol), crude 24b (1.20 g, 4.16 mmol) and pot- was treated as in 25c to give 26 (93 mg, 0.36 mmol,
assium hydroxide (1.17 g, 20.8 mmol) was treated as in 24.6%) as a yellow needle. 1H NMR (acetone-d6,
10c to give 24 (0.11 g, 0.39 mmol, 15.9%) as a yellow 300 MHz) δ: 12.82 (s, 1H), 7.83 (d, J l 8.4, 1H), 6.41 (d,
needle. 1H NMR (acetone-d6) δ: 13.33 (s, 1H), 8.01 (d, J l 8.4, 1H), 6.32 (d, J l 2.1, 1H), 7.73 (dd, J l 2.1, 8.4,
J l 8.4, 1H), 8.18 (d, J l 15.3, 1H), 7.81 (d, J l 15.3, 1H), 6.7–6.8 (m, 1H), 7.02 (dd, J l 2.1, 8.4, 1H), 7.15
1H), 7.22 (d, J l 8.4, 1H), 7.76 (dd, J l 2.1, 8.4, 1H), (dd, J l 2.1, 8.4, 1H), 3.23 (t, J l 7.2, 2H), 2.94 (t, J l
6.30 (d, J l 8.4, 1H), 6.38 (d, J l 2.1, 1H). 13C NMR 7.2, 2H). 13C NMR (acetone-d6): δ 117.8 (C-1h), 158.1
(acetone-d6): δ 116.9 (C-1h), 160.3 (C-2h), 103.0 (C-3h), (C-2h), 101.9 (C-3h), 162.8 (C-4h), 107.9 (C-5h), 131.7 (C-
165.5 (C-4h), 108.3 (C-5h), 132.5 (C-6h), 187.1 (CꢀO), 6h), 198.1 (CꢀO), 42.7 (C-α), 19.9 (C-β), 127.4 (C-1),
123.3 (C-α), 142.7 (C-β), 116.1 (C-1), 143.6 (C-2), 131.6 156.7 (C-2), 115.4 (C-3), 127.1 (C-4), 121.0 (C-5), 128.7
(C-3), 145.1 (C-4), 109.6 (C-5), 121.6 (C-6). TLC, Rf (C-6). TLC, Rf 0.62 (5:3 hexane\EtOAc); IR (KBr)
1
1
−
−
+
0.35 (1:3 hexane\EtOAc); IR (KBr) 3422, 1625 cm ; 3477, 1649 cm ; EI-MS (70 eV) m\z 256 (M , 27), 137
+
EI-MS (70 eV) m\z 288 (M , 21), 137 (100), 179 (62), (100), 149 (54), 121 (14), 107 (26); HR-MS (EI) exact
+
151 (32), 109 (20); HR-MS (EI) exact mass for C15H12O6 mass for C15H14O4 (M ) 258.0892, found 258.0870.
+
(M ) 288.0634, found 288.0629.
2,4,2h,4h-Tetrahydroxydihydrochalcone (27)
A mixture of 2,4,2h,4h-tetra(methoxymethoxy)chalcone
(0.5 g, 1.24 mmol) and 10% palladium on charcoal
General procedure for the preparation of
2h-hydroxydihydrochalcone (25–27)
(5 mg)wastreatedasin25ctogive27(0.18 g, 0.65 mmol,
52.8%) as a yellow needle. 1H NMR (acetone-d6,
4,2h,5h-Trihydroxydihydrochalcone (25)
A solution of 4,2h,5h-Tris(methoxymethoxy)chalcone 300 MHz) δ: 12.84 (s, 1H), 7.82 (d, J l 8.7, 1H), 6.33 (d,
(0.5 g, 1.45 mmol) in ethyl hydroxide (20 mL) was J l 8.7, 1H), 6.23 (d, J l 8.7 1H), 6.93 (d, J l 8.7, 1H),
hydrogenated at 1 atm for 4 h in the presence of 10% 6.41 (s, 1H), 3.16 (t, J l 7.2, 2H), 2.87 (t, J l 7.2, 2H).
palladium on charcoal (5 mg) at room temperature. The 13C NMR (acetone-d6): δ117.2 (C-1h), 158.8 (C-2h),
catalyst was removed by filtration through Celite and 102.8 (C-3h), 163.1 (C-4h), 108.2 (C-5h), 131.4 (C-6h),
washed with ethyl hydroxide (100 mL). The combined 197.6 (CꢀO), 41.8 (C-α), 19.7 (C-β), 120.0 (C-1), 158.1
filtrate was evaporated to afford crude 4,2h,5h-Tris- (C-2), 102.8 (C-3), 155.9 (C-4), 108.2 (C-5), 130.7 (C-6).
(methoxymethoxy)-dihydrochalcone (25c), which was TLC, Rf 0.46 (5:3 hexane\EtOAc); IR (KBr) 3447,
1
−
+
dissolved in methyl hydroxide (20 mL). To this solution 1644 cm ; EI-MS (70 eV) m\z 272 (M , 41), 165 (29),
was added 2 mL 3  HCl. After 3 h, the reaction was 138 (29), 137 (100), 123 (14); HR-MS (EI) exact mass
+
concentrated and diluted with ethyl acetate, washed for C15H14O5 (M ) 274.0841, found 274.0829.

662
Soon Sung Lim et al
+
(14), 75 (21); HR-MS (EI) exact mass for C15H12O4 (M )
General procedure for the preparation of
flavanone (28–30)
256.0736, found 256.0718.
6,4h-Dihydroxyflavanone (28)
7,2h,4h-Trihydroxyflavanone (30)
2h-Hydroxy-5h-methoxymethoxyacetophenone (0.98 g,
5 mmol) and 4-methoxymethoxy-benzaldehyde (0.82 g,
5 mmol) were added to a mixture of H3BO3 (0.46 g,
7.5 mmol), piperidine (0.11 g, 1.25 mmol) and SiO2
(2.5 g) in DMF (20 mL). The content was stirred and
heated at 120mC under nitrogen for 6–12 h. The mixture
was then cooled to room temperature and diluted with
acetone (20 mL) and filtered. The SiO2 was rinsed with
acetone (10 mL) three times. The acetone solution was
combined with the filtrate and evaporated at reduced
pressure to dryness to afford crude 6,4h-di(methoxy-
methoxy)flavanone (28c). To a stirred solution of 28c
(1.07 g, 3.1 mmol) in 20 mL methanol was added 5 mL
3  HCl. After 3 h, the reaction was concentrated and
diluted with ethyl acetate, washed with water and brine,
dried over magnesium sulfate, filtered and evaporated.
This crude solid was purified by column chro-
matography (silica gel, eluted with 38% ethyl acetate in
hexane) to 28 (0.56 g, 2.19 mmol, 43.8%) as a yellow
needle. 1H NMR (acetone-d6, 300 MHz) δ: 7.98 (s, 1H),
6.88 (d, J l 8.7, 1H), 6.78 (d, J l 8.7, 1H), 7.37 (d, J l
8.7, 2H), 7.05 (d, J l 8.7, 2H), 5.41 (dd, J l 2.7, 13.2,
1H), 3.39 (dd, J l 13.2, 16.8, 1H), 3.32 (dd, J l 2.7,
16.8, 1H). 13C NMR (acetone-d6): δ 72.9 (C-2), 48.8
(C-3), 197.6 (C-4), 116.4 (C-5), 148.8 (C-6), 120.7 (C-7),
115.5 (C-8), 133.5 (C-1h), 128.7 (C-2h), 115.9 (C-3h),
156.2 (C-4h), 115.9 (C-5h), 128.7 (C-6h). TLC, Rf 0.49
A mixture of 4h-methoxymethoxy-2h-hydroxyaceto-
phenone (0.5 g, 2.56 mmol) and 2,4-di(methoxy-
methoxy)-benzaldehyde (0.57 g, 2.56 mmol) was treated
as in 28c to give 30 (0.1 g, 0.37 mmol, 14.3%) as a
yellow needle. 1H NMR (acetone-d6, 300 MHz) δ: 7.99
(d, J l 8.7, 1H), 6.40 (d, J l 8.7, 1H), 6.33 (s, 1H), 7.71
(d, J l 8.7, 1H), 7.34 (d, J l 8.7, 1H), 6.85 (s, 1H), 5.75
(dd, J l 2.4, 12.8, 1H), 3.42 (dd, J l 12.8, 17.1, 1H),
2.89 (dd, J l 2.4, 17.1, 1H). 13C NMR (acetone-d6): δ
62.7 (C-2), 49.1 (C-3), 197.8 (C-4), 130.6 (C-5), 107.2
(C-6), 162.3 (C-7), 101.3 (C-8), 120.7 (C-1h), 157.5 (C-2h),
103.1 (C-3h), 157.6 (C-4h), 108.5 (C-5h), 130.1 (C-6h). 13C
NMR (acetone-d6): δ 168.8 (C-2), 94.9 (C-3), 187.0
(C-4), 117.6 (C-5), 152.0 (C-6), 122.2 (C-7), 118.9 (C-8),
127.5 (C-1h), 127.6 (C-2h), 115.6 (C-3h), 156.5 (C-4h),
115.6 (C-5h), 127.6 (C-6h). TLC, Rf 0.43 (5:3 hexane\
1
−
EtOAc); IR (KBr) 1622, 1221 cm ; EI-MS (70 eV) m\z
+
272 (M , 36), 137 (100), 124 (72), 107 (21), 100 (20)
(Fukai et al 1996).
General procedure for the preparation of
flavone (31–33)
6,4h-Dihydroxyflavone (31)
To a solution of 2h-hydroxy-4,5h-di(methoxymethoxy)-
chalcone (400 mg, 1.16 mmol) in DMSO (5 mL) at
150mC was added iodine (15 mg, 0.06 mmol) with stir-
ring. After being stirred for 0.5 h, the mixture was
poured into a cold solution of Na2S2O3 (1.0 g) and KOH
(1.0 g) in water (50 mL). The resulting solution was
dilutedwith ethylacetate. Theorganic phasewas washed
with brine, dried over anhydrous magnesium sulfate
and concentrated. The compound 31c was obtained in
1
−
(5:3 hexane\EtOAc); IR (KBr) 1611, 1219 cm ; EI-
+
MS (70 eV) m\z 256 (M , 29), 137 (100), 107 (28), 93
+
(17), 75 (14); HR-MS (EI) exact mass for C15H12O4 (M )
256.0736, found 256.0719.
7,2h-Dihydroxyflavanone (29)
A mixture of 4h-methoxymethoxy-2h-hydroxyaceto- 50.8% isolated yield. To a stirred solution of 31c (0.20 g,
phenone (0.59 g, 3.03 mmol) and 2-methoxymethoxy- 0.59 mmol) in 20 mL methanol was added 5 mL 3 
benzaldehyde (0.5 g, 3.03 mmol) was treated as in 28c to HCl. After 3 h, the reaction was concentrated and
give 29 (0.26 g, 1.03 mmol, 34.1%) as a yellow needle. diluted with ethyl acetate, washed with water and brine,
1H NMR (acetone-d6, 300 MHz) δ: 8.06 (d, J l 8.7 dried over magnesium sulfate, filtered and evaporated.
1H), 6.43 (dd, J l 2.1, 8.7, 1H), 6.31 (d, J l 8.4, 1H), This crude solid was purified by column chroma-
7.85 (d, J l 2.1, 1H), 6.8–7.0 (m, 1H), 7.15 (dd, J l 2.1, tography (silica gel, eluted with 38% ethyl acetate in
8.4, 1H), 7.27 (dd, J l 2.1, 8.4, 1H), 5.78 (dd, J l 2.7, hexane) to 31 (65 mg, 0.25 mmol, 22.8%) as a yellow
12.3, 1H), 3.00 (dd, J l 12.3, 17.1, 1H), 2.81 (dd, J l needle. 1H NMR (acetone-d6, 300 MHz) δ: 7.93 (d, J l
2.7, 17.1, 1H). 13C NMR (acetone-d6): δ 62.3 (C-2), 48.9 2.1, 1H), 6.98 (d, J l 8.7, 1H), 6.65 (dd, J l 2.1, 8.7,
(C-3), 196.4 (C-4), 130.1 (C-5), 106.8 (C-6), 161.7 (C-7), 1H), 6.54 (s, 1H), 7.58 (d, J l 8.4, 2H), 7.29 (d, J l 8.4,
101.1 (C-8), 128.1 (C-1h), 156.1 (C-2h), 115.1 (C-3h), 2H). 13C NMR (acetone-d6): δ 167.9 (C-2), 93.6 (C-3),
128.8 (C-4h), 121.3 (C-5h), 128.1 (C-6h). TLC, Rf 0.36 189.2 (C-4), 117.6 (C-5), 152.0 (C-6), 122.2 (C-7), 118.9
1
−
(5:3 hexane\EtOAc); IR (KBr) 1612, 1227 cm ; EI- (C-8), 127.5 (C-1h), 127.6 (C-2h), 114.6 (C-3h), 156.5 (C-
+
MS (70 eV) m\z 256 (M , 44), 137 (100), 107 (36), 93 4h), 115.6 (C-5h), 127.1 (C-6h). TLC, Rf 0.49 (1:3 hexane\

663
Synthesis of flavonoids and their effects on diabetic complications
1
−
EtOAc); IR (KBr) 1601, 1221 cm ; EI-MS (70 eV) m\z was purified by column chromatography (silica gel,
+
254 (M , 24), 137 (100), 121 (46), 107 (72), 93 (49) (Singh eluted with 38% ethyl acetate in hexane) to 34 (52.8 mg,
& Singh 1985).
0.19 mmol, 48.7%) as a yellow needle. 1H NMR
(acetone-d6, 300 MHz) δ: 7.78 (d, J l 2.1, 1H), 6.90 (d,
J l 8.4, 1H), 6.79 (d, J l 8.4, 1H), 7.33 (d, J l 8.4, 2H),
7,2h-Dihydroxyflavone (32)
2h-Hydroxy-2,4h-di(methoxymethoxy)chalcone (0.5 g, 7.04 (d, J l 8.4, 2H). 13C NMR (acetone-d6): δ 134.1 (C-
1.45 mmol) was treated as in 31c to give 32 (78.7 mg, 2), 122.1 (C-3), 187.7 (C-4), 117.6 (C-5), 153.4 (C-6),
1
0.31 mmol, 21.6%) as a pale brown needle. H NMR 123.6 (C-7), 117.9 (C-8), 126.9 (C-1h), 127.3 (C-2h), 114.9
(acetone-d6, 300 MHz) δ: 7.98 (d, J l 8.4, 1H), 7.14 (s, (C-3h), 155.9 (C-4h), 114.9 (C-5h), 127.3 (C-6h). TLC, Rf
1
−
1H), 6.51 (d, J l 8.4, 1H), 7.61 (d, J l 8.4, 1H), 6.9–7.0 0.36 (1:3 hexane\EtOAc); IR (KBr) 1603, 1214 cm ;
+
(m, 1H), 7.13 (dd, J l 2.1, 8.4, 1H), 7.38 (d, J l 8.4 1H), EI-MS (70 eV) m\z 270 (M , 19), 137 (100), 121 (29),
6.56 (s, 1H). 13C NMR (acetone-d6): δ 168.8 (C-2), 94.9 109 (19), 93 (18); HR-MS (EI) exact mass for C15H11O5
+
(C-3), 187.0 (C-4), 131.8 (C-5), 110.4 (C-6), 163.8 (C-7), (M ) 271.0606, found 271.0639.
104.7 (C-8), 122.1 (C-1h), 155.0 (C-2h), 115.6 (C-3h),
129.1 (C-4h), 121.0 (C-5h), 128.4 (C-6h). TLC, Rf 0.44
7,2h-Dihydroxyflavonol (35)
(1:3 hexane\EtOAc); IR (KBr) 1602, 1219 cm–1 ; EI-
2h-Hydroxy-2,4h-di(methoxymethoxy)chalcone (0.5 g,
1.45 mmol) was treated as in 34c to give 35 (0.23 g,
1
0.84 mmol, 57.8%) as a pale brown needle. H NMR
+
MS (70 eV) m\z 254 (M , 46), 137 (100), 121 (24), 107
(19), 93 (41) (Ahmad et al 1991).
(acetone-d6, 300 MHz) δ: 7.87 (d, J l 8.4, 1H), 6.43 (d,
J l 8.4, 1H), 6.38 (s, 1H), 7.73 (d, J l 8.4, 1H), 6.8–7.0
(m, 1H), 6.64 (dd, J l 2.1, 8.4, 1H), 7.13 (d, J l 8.4 1H).
13C NMR (acetone-d6): δ 133.5 (C-2), 122.6 (C-3), 187.0
(C-4), 130.6 (C-5), 110.2 (C-6), 162.9 (C-7), 104.2 (C-8),
121.4 (C-1h), 154.5 (C-2h), 115.1 (C-3h), 128.4 (C-4h),
120.7 (C-5h), 127.6 (C-6h). TLC, Rf 0.46 (1:6 hexane\
7,2h,4h-Trihydroxyflavone (33)
2h-Hydroxy-2,4,4h-Tris(methoxymethoxy)chalcone
(0.5 g, 1.24 mmol) was treated as in 31c to give 33
(0.14 g, 0.54 mmol, 43.2%) as a yellow needle. 1H NMR
(acetone-d6, 300 MHz) δ: 8.05 (d, J l 8.4, 1H), 6.41 (dd,
J l 2.1, 8.4, 1H), 6.34 (d, J l 2.1, 1H), 7.57 (d, J l 8.7,
1H), 6.54 (dd, J l 2.1, 8.4, 1H), 6.51 (d, J l 2.1, 1H),
6.32 (s, 1H). 13C NMR (acetone-d6): δ 132.4 (C-2), 121.9
(C-3), 189.0 (C-4), 131.4 (C-5), 111.6 (C-6), 163.1 (C-7),
103.6 (C-8), 114.7 (C-1h), 156.4 (C-2h), 102.8 (C-3h),
157.9 (C-4h), 108.2 (C-5h), 129.0 (C-6h). TLC, Rf 0.39
1
−
EtOAc); IR (KBr) 1601, 1219 cm ; EI-MS (70 eV) m\z
+
270 (M , 41), 137 (100), 121 (37), 109 (24), 93 (31); HR-
+
MS (EI) exact mass for C15H11O5 (M ) 271.0606, found
271.0612.
1
−
(1:3 hexane\EtOAc); IR (KBr) 1602, 1210 cm ; EI-
+
MS (70 eV) m\z 270 (M , 29), 137 (100), 123 (24), 109
Results and Discussion
+
(16), 93 (29); HR-MS (EI) exact mass for C15H10O5 (M )
270.0528, found 270.0512.
The chemical structures of the synthesized flavonoid
derivatives, their yields and the physicochemical data
are shown in Materials and Methods. Known flavonoids
are indicated by supplementary reference and flavonoids
without reference are new compounds (Table 1). The
General procedure for the preparation of
flavonol (34, 35)
6,4h-Dihydroxyflavonol (34)
structures of new flavonoid derivatives were elucidated
1
2h-Hydroxy-4,5h-di(methoxymethoxy)chalcone(144 mg, by H and 13C NMR spectra. Their high-resolution
+
0.4 mmol) was dissolved in ethanol (1.5 mL). Sodium mass spectra (M ) were within p0.9 millimass unit of
hydroxide (1 , 0.4 mL) and hydrogen peroxide (0.5 g) the calculated values. The geometry of chalcones was
were added, and the mixture was heated at 90mC for proved trans by 1H NMR spectrum in which the
35 min. The cooled mixture was diluted with water coupling constant between the two vinylic protons ap-
(5 mL) and acidified with hydrochloric acid (1 ). A pearing at 6.94–7.95 and 7.18–8.22 ppm were approxi-
precipitate was collected and recrystallized from meth- mately 15 Hz.
anol. The compound 34c was obtained in 60.0% isolated
Structure–activity relationships among flavonoids
yield. To a stirred solution of 34c (86.4 mg, 0.24 mmol) were estimated by their effects on rat lens aldose re-
in 20 mL methanol was added 5 mL 3  HCl. After 3 h, ductase using -glyceraldehyde as a substrate and by
the reaction was concentrated and diluted with ethyl their free radical scavenging effect on DPPH (Table 1).
acetate, washed with water and brine, dried over magne- The inhibitory activities on the enzyme were expressed
sium sulfate, filtered and evaporated. This crude solid as percent inhibition at 1.0 µ and as IC50 values. The

664
Soon Sung Lim et al
′
5
′
′
4
6
R2
R2
R1
R2
′
8
5
5
5
O
O
O
O
′
7
′
′
4
6
4
R2
3
3
6
R1
′
2
R1
R2
R1
R1
OH
6
′
3
2
O
O
OH
O
OH
O
1 – 24
25 – 27
28 – 30
31 – 33
34, 35
Table 1 The physicochemical and biological data of flavonoid derivatives.
Compound
R1
R2
mp (mC) (lit. mp)c
% yield
Formula
Rat lens aldose
reductasea IC50 (µ)
DPPHb IC50 (µ)
1
H
4-OH
160–161 (162–162.5) 22.9d
C15H12O3
C15H12O4
C15H12O4
C16H14O4
C16H14O4
C16H14O4
C16H14O3
4.25
0.40
5.39
5.24
3.18
3.27
4.38
1.01
2.15
1.01
0.38
0.93
1.12
3.21
0.81
1.15
1.05
0.45
1.21
0.91
0.40
0.16
0.58
0.75
" 10.0
0.62
2.95
" 10.0
1.66
2.29
5.25
" 10.0
0.35
" 10.0
" 10.0
64.8p4.3
54.7p3.2
20.7p3.6
85.4p6.9
88.5p5.1
31.5p2.5
32.8p2.6
79.2p9.2
88.9p4.6
85.6p5.8
48.2p6.2
85.9p5.9
145.9p9.8
198.6p6.9
230.1p12.3
189.5p10.5
89.6p9.5
7.40p0.8
150.6p10.4
187.6p6.8
190.2p7.9
38.2p3.2
20.5p2.4
30.4p3.6
10.2p1.2
11.8p0.9
12.7p1.1
87.8p0.7
97.5p8.7
107.5p0.9
85.9p8.7
121.5p10.2
98.2p7.8
13.8p8.5
32.5p2.7
2
4h-OH
5h-OH
4h-OCH3
5h-OCH3
6h-OCH3
5h-CH3
5h-Cl
4-OH
188–190 (187–188)
191–192 (191–192)
150–153 (151–154)
158–160
17.4d
21.1d
24.4e
24.1e
28.9e
19.8d
17.3d
22.5d
22.9d
17.0d
24.1e
35.8e
8.74d
16.0d
25.2d
11.3e
21.5e
33.6e
30.6e
29.4e
28.6e
23.9e
15.9e
43.5e
24.6e
52.8d
43.8f
34.1d
14.3d
22.8f
21.6f
43.2f
48.7e
57.8e
3
4-OH
4
4-OH
5
4-OH
6
4-OH
175–177 (171–174)
96–97
7
4-OH
8
4-OH
155–157
C15H11O3Cl
C15H11O3Br
C15H12O3
C15H12O4
C16H14O4
C15H11O3Br
C15H11O5N
C16H14O3
C17H17O3N
C17H16O5
C15H12O5
C15H10O3Cl2
C16H13O4F
C17H16O3
C15H12O5
C15H12O5
C15H12O6
C15H14O4
C15H14O4
C15H14O5
C15H12O4
C15H12O4
C15H12O5
C15H10O4
C15H10O4
C15H10O5
C15H11O5
C15H11O5
9
5h-Br
4-OH
135–137
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
4h-OH
6-OH
7-OH
7-OH
6-OH
7-OH
7-OH
6-OH
7-OH
H
145–148 (149–150)
194–196 (193–194)
130–133
2-OH
4-OCH3
4-Br
162–164
4-NO2
219–222 (223–225)
137–139
4-CH3
4-N(CH3)2
3,4-diOCH3
3,4-diOH
3,4-diCl
3-F,4-OCH3
2,4-diCH3
2,4-diOH
2,5-diOH
2,3,4-triOH
4-OH
144–146
112
214–216 (211–213)
103–107
146–148
110–112
" 320 (" 320)
156–158
164–169
214–217
2-OH
261–267
2,4-diOH
4h-OH
2h-OH
2h,4h-diOH
4h-OH
2h-OH
2h,4h-diOH
4h-OH
2h-OH
295–297
236–238
202–204
221–223 (225–226)
" 320 (" 320)
320 (" 320)
140–142
203–206
230–232
aRat lens aldose reductase activities were determined by measuring the decrease in absorption of NADPH at 340 nm in a reaction mixture
containing 100 m phosphate buffer (pH 6.3), 0.3  ammonium sulphate, 1 m EDTA, 0.2 m NADPH, and 10 m -glyceraldehyde in a
final volume of 1 mL. bIn microwells, 100 µL of an aqueous solution of the sample (control: 100 µL distilled water) was added to an ethanolic
solution of DPPH (60 µ). Seven concentrations were prepared for each sample. After mixing gently and leaving to stand for 30 min at room
c
temperature, the optical density was determined using a microplate reader at 517 nm. Parenthesis numbers are literature melting points of
known flavonoids. Solvent used for recrystallization: dmethanol, ecyclohexane–ethyl acetate, fethanol–water.

665
Synthesis of flavonoids and their effects on diabetic complications
Table 2 Bodyweight and blood glucose concentrations of diabetic
rats.
rat lens aldose reductase assay demonstrated that a
hydroxyl group in position C-4h was important to aldose
reductase inhibition activity. This finding led to the
study of the activity of compounds 1–9. While com-
pound 2, which bears only the 4h-hydroxyl, was active
(IC50 l 0.40 µ), the corresponding methoxylated
compounds 4 and 3 (5h-hydroxyl), hydroxylated in a
different position, were considerably less active (IC50 l
5.24 and 5.39 µ, respectively) on rat lens aldose re-
ductase.
Group
Bodyweight (g)
Blood glucose
1
−
(mg dL
)
Normal (6)
289p8
179p6
185p9
192p5
175p9
191p7
176p8
174p6
123p3
Diabetic control (7)
Diabeticjepalrestat (6)
Diabeticj2 (6)
Diabeticj11 (6)
Diabeticj18 (6)
Diabeticj21 (6)
Diabeticj22 (6)
459p23
469p15
453p26
476p20
492p19
453p23
468p10
Given that the pKa value (! 7.47) (Rastelli et al 2000)
of compound 2 approached the pH of the enzymatic
assay, these compounds probably act in their anionic
forms, in line with the general view (Bors & Saran 1987)
regarding the importance of the presence of anionic
forms on the inhibition of aldose reductase. In support
of this hypothesis, the methoxylated compound 4, in
which proton dissociation was prevented, was found to
be much less active than 2.
Values are meanps.e.m. The number of animals in each group is
given in parentheses.
to the fundamental significance of this structural ar-
2h,4h-Dihydroxychalcone derivatives (10–24), except rangement. These results suggested that an isolated
compound 14, with a 4-nitro group in the B-ring when hydroxyl group on the B ring made no contribution to
used at a concentration of 10 6  reduced rat lens aldose the free radical scavenging activity. Moreover, when the
−
reductase activity by more than 45%. The compounds free hydroxyl group was methoxylated as it was in
possessing the highest aldose reductase inhibitory ac- compounds 5 or 17, the scavenging activity was signifi-
tivity were 2,4,2h,4h-tetrahydroxychalcone (22, IC50 l cantly decreased.
0.16 µ), 2,2h,4h-trihydroxychalcone (11, IC50 l
As shown in the DPPH assay (Table 1), 3,4,2h,4h-
0.38 µ), 2h,4h-dihydroxy-2,4-dimethylchalcone (21, tetrahydroxychalcone (18) acted as a free radical scav-
IC50 l 0.4 µ) and 3,4,2h,4h-tetrahydroxychalcone (18, enger with a potency (IC50 l 7.40p0.8 µ) similar to
IC50 l 0.45 µ) for -glyceraldehyde, which were α-tocopherol, a chain-breaking antioxidant.
slightly less potent than epalrestat (IC50 l 0.08 µ), a
In the case of other the flavonoids, compounds 25, 26
known potent aldose reductase inhibitor. At present, and27(reductionfrom3, 11and22, respectively)exerted
only epalrestat, which reached the Japanese market in significant scavenging effect on DPPH free radical, while
1992, is available for clinical trials (Costantino et al the inhibitory activities on aldose reductase were insig-
1999).
nificant. Compounds 28–30, flavanones, showed weak
Antioxidant or free radical scavenging activity of activity in both assay systems. In the case of flavones 31
flavonoids has been demonstrated to be related to the and 33 (not 32) the inhibition on aldose reductase
number and position of free hydroxyl groups, which showed a similar activity so long as the substitution
could act through their hydrogen donating capability pattern was the same, but radical-scavenging effect
(Bors & Saran 1987; Cotelle et al 1992). The DPPH showed weak activity. The flavonols 34 and 35 exhibited
system is a stable radical-generating procedure (Hatano slightly stronger radical-scavenging effects on DPPH.
et al 1989). It can accommodate a large number of This showed that the hydroxyl group at C-3 in flavone
samples in a short time and is sensitive enough to detect and the saturation of α, β bond of a compound enhanced
active principles at low concentrations, and so it was its scavenging effects on free radical DPPH (Dziedzic et
used in this study for primary screening of the free- al 1985; Yokozawa et al 1998).
radical-scavenging activities of 24 synthetic chalcones.
An in-vivo study showed the efficacy of synthetic
Unfortunately, the chalcones tested showed a weak free chalcones, in comparison with epalrestat, on sorbitol
radical scavenging activity except for 3, 18, 23 which accumulation in the tissues of diabetic rats. The diabetes
had an o-dihydroxy or hydroquinone moiety. The pres- was confirmed by measurement of blood glucose. Al-
ence of a single hydroxy group in the B ring of com- though there were no significant differences in body
pounds 1, 2, 4–9 and 11 in place of the o-dihydroxy (18) weights or blood glucose concentrations between the
and hydroquinone (23) structure caused a marked re- rats treated with synthetic chalcones and diabetic con-
duction in the free radical scavenging activity, attesting trol rats (Table 2), the mean sorbitol contents of the

666
Soon Sung Lim et al
R
HO
2
4-OH
11 2-OH
R
18 3,4-diOH
21 3,4-diOCH3
22 2,4-diOH
OH
O
Table 3 The effects of synthetic chalcones and epalrestat on the accumulation of sorbitol in the tissues of
diabetic rats.
Group
RBC (nmol
Sciatic nerve (nmol
1
Lens (nmol
(mg wet wt)
1
a
a
1 a
−
−
−
(g haemoglobin)
)
(mg wet wt) ) [%
inhibition]
)
[% inhibition]
[% inhibition]
Normal (6)
74.5p6.5
0.14p0.01
0.2p0.01
Diabetic control (7)
Diabeticjepalrestat (6)
Diabeticj2 (6)
Diabeticj11 (6)
Diabeticj18 (6)
Diabeticj21 (6)
Diabeticj22 (6)
163.6p5.7FFF
0.47p0.03FFF
18.9p2.3FFF
114.3p7.3 [55.3]**
127.5p4.7 [40.5]**
145.3p8.0 [20.5]
115.5p6.8 [54.0]**
124.0p9.8 [44.4]**
113.7p6.6 [56.0]**
0.38p0.02 [27.2]*
0.29p0.02 [54.5]**
0.28p0.04 [57.2]**
0.24p0.02 [69.7]***
0.42p0.03 [15.1]
0.30p0.03 [51.5]*
15.8p1.2 [16.6]
16.5p1.3 [12.8]
15.1p1.5 [20.3]
14.7p1.3 [22.5]*
14.6p1.2 [23.0]*
15.1p1.0 [20.3]
Animals were given the vehicle alone, synthetic compounds (2, 12, 19, 22, 23) or epalrestat via intragastric
1
−
tubing twice a day at a dose of 75 mg kg \day for two weeks. The number of animals in each group is given
in parenthesis. Meanps.e.m. % Inhibition l (the sorbitol contents of control – the sorbitol contents of
a
sample)\( the sorbitol contents of control – the sorbitol contents of normal)i100. *P ! 0.05, **P ! 0.01,
***P ! 0.001 compared with control. FFFP ! 0.001 compared with normal.
RBC, sciatic nerves and lenses were observed to alter sciatic nerves. In the lens, compounds 2, 11, 18, 21, 22
significantly, as shown in Table 3. In diabetic control and epalrestat decreased sorbitol contents by 12.8, 20.3,
rats, the sorbitol contents of the RBC (163.6p5.7 nmol 22.5, 23.0, 20.3 and 16.6%, respectively.
1
−
(g haemoglobin) , P ! 0.001 compared with normal)
Previous reports suggested that glucose-induced oxi-
was much higher than in normal rats (74.5p6.5 nmol (g dative stress played a causative role in induction of
1
haemoglobin) ). In diabetic rats treated with chalcones aldose reductase in sensitive tissues exposed to long-
−
(2, 11, 18, 21, 22) or epalrestat, the accumulation of term hyperglycaemia (Tawata et al 1992) and that
sorbitol in the RBC was significantly inhibited by 40.5% increased oxidant production by metal-catalysed glu-
(P ! 0.01 compared with control), 20.5%, 54.0% (P ! cose oxidation may be an important mechanism in
0.01), 44.4% (P ! 0.01), 56.0% (P ! 0.01), and 55.3% activation of aldose reductase (Ou et al 1996). Therefore,
(P ! 0.01), respectively. The sorbitol content of the antioxidant and selective metal-chelating agents would
sciatic nerves in diabetic rats was shown to be 0.47 nmol be a useful tool in the experimental approach for the
(mg wet wt) 1 (P ! 0.001 compared with normal), which prevention of induction and activation of aldose re-
was significantly higher than that of the normal rats ductase.
1
(0.14 nmol (mg wet wt) ). Following treatment with
−
−
The abilities of the chalcones tested as transition-
chalcones 2, 11 and 22, the accumulation of sorbitol in metal-chelators in-vitro are shown in Figure 1. The
the sciatic nerves was significantly inhibited by 54.5% chalcones 11, 18 and 22 enhanced partitioning of copper
(P ! 0.01 compared with diabetic control), 57.2% (P ! ion into n-octanol by 1.43p0.08, 2.32p0.09 and
0.01) and 51.5% (P ! 0.05), respectively. Compounds 1.26p0.05 µ, respectively. They were more effective in
2, 11 and 22 except 21 (15.1%) suppressed the ac- partitioning copper ions into n-octanol than the hydro-
cumulation of sorbitol in sciatic nerves stronger than philic metal chelator EDTA (1.25p0.05 µ). Among
epalrestat (27.2%, P ! 0.05 compared with control). the five chalcones, 2h,3,4,4h-tetrahydroxychalcone (18),
Among the five chalcones tested, 3,4,2h,4h-tetra- with o-dihydroxy, was the highest transition-metal-
hydroxychalcone (18) (69.7%, P ! 0.001) was the most chelator and free radical scavenger (Figure 2 and
potent suppressor of the accumulation of sorbitol in Table 1).

667
Synthesis of flavonoids and their effects on diabetic complications
T., Chin, M., Mitsuhashi, H. (1990) Isoliquiritigenin: a new aldose
reductase inhibitor from Glycyrrhizae radix. Planta Med. 57:
254–258
2.5
2.0
1.5
1.0
Beyer-Mears, A., Cruz, E. (1985) Reversal of diabetic cataract by
sorbinil, an aldose reductase inhibitor. Diabetes 34: 15–21
Bhartiya, H. P., Gupta, P. C. (1982) A chalcone glycoside from the
flowers of Adhatoda vasica. Phytochemistry 21: 247
Bors, W., Saran, M. (1987) Radical scavenging by flavonoid antioxi-
dants. Free Radic. Res. Commun. 2: 289–294
Carper, D. A., Sun, J. K., Iwata, T., Zigler, J. S., Ibaraki, N., Lin, L.-
R., Reddy, V. (1999) Oxidative stress induces differential gene
expression in a human lens epithelial cell line. Invest. Ophthalmol.
Vis. Sci. 40: 400–406
0.5
0.0
Cheng, Z., Kuo, S., Chan, S., Ko, F., Teng, C. (1998) Antioxidant
properties of butein isolated from Dalbergia odorifera. Biochim.
Biophys. Acta 1392: 291–299
2
11 18 21 22
Costantino, L., Rastelli, G., Vianello, P., Cignarella, G., Barlocco, D.
(1999) Diabetes complications and their potential prevention:
aldose reductase inhibition and other approaches. Med. Res. Rev.
19: 3–23
Treatment
2
+
Figure 2 Effect of chalcones on Cu partitioning into n-octanol.
Stock solutions of compounds (all at 2.5 m) were prepared in PBS in
the presence of 1 m CuSO4. Samples (1 mL) of the compounds were
mixed with n-octanol (2 mL) and were shaken for 10 min. After
Cotelle, N., Bernier, J. L., Henichart, J. P., Catteau, J. P., Gaydou,
E., Wallet, J. C. (1992) Scavenger and antioxidant properties of ten
synthetic flavones. Free Radic. Biol. Med. 13: 211–219
Delle-Monache, G., Cristina-De-Rosa, M., Scurria, R., Vitali, A.,
Cuteri, A., Monacelli, B., Pasqua, G., Botta, B. (1995) Comparison
between metabolite productions in cell culture and in whole plant of
Maclura pomifera. Phytochemistry 39: 575–580
2
+
centrifugationat1000g, Cu content inthe organiclayerwas analysed
by atomic absorption spectrophotometry. Data represent the meanp
s.d. of triplicate samples. *P ! 0.05, **P ! 0.01 compared with
control). Control, vehicle alone; EDTA, positive control.
Dubowski, M. K. (1962) Methods for body glucose determination.
Clin. Chem. 8: 215–253
Dziedzic, S. Z., Hudson, B. J. F., Barnes, G. (1985) Polyhydroxy-
dihydrochalcones as antioxidants for lard. J. Agric. Food Chem. 33:
244–246
3,4,2h,4h-Tetrahydroxychalcone (18, butein) has
been isolated from Dalbergia odorifera. It has been
reported to be an inhibitor of xanthine oxidase and
exhibit various antioxidant effects such as free radical
scavenging, metal ion chelation and protection from
lipid peroxidation in rat brain and human low density
lipoprotein (Cheng et al 1998).
Fukai, T., Yue-Hu, P., Nomura, T., Chun-Quan, X., Li-Jun, W.,
Ying-Jie (1996) Components of the root bark of Morus cathayana.
1. Structures of five new isoprenylated flavonoids, sanggenols A-E
and a diprenyl-2-arylbenzofuran, mulberrofuran. Heterocycles 43:
425–436
Geissman, T. A., Clinton, R. O. (1946) Flavanones and related com-
pounds. 1. The preparation of polyhydroxychalcones and -flava-
nones. J. Am. Chem. Soc. 68: 697–700
In conclusion, from in-vitro and in-vivo data there
are strong indications that the antioxidant effect by
some synthetic chalcone derivatives, as well as inhibition
of aldose reductase, plays an important role in the
inhibition of the accumulation of sorbitol in the tissues
in streptozotocin-induced diabetic rats. As a result, we
havepresented somenew chalconederivativesas a group
of potent sorbitol suppressors in the tissues of diabetic
rats, in which 3,4,2h,4h-tetrahydroxychalcone (18) is
the most promising compound for further investigation.
Grasselli, J. G., Ritchey, W. M. (1975) Atlas of Spectral Data and
Physical Constants for Organic Compounds. 2nd edn, CRC, Cleve-
land, Ohio, pp 3
Handelsman, D. J., Turtle, J. R. (1981) Clinical trial of an aldose
reductase inhibitor in diabetic neuropathy. Diabetes 30 : 459–464
Hatano, T., Edamatsu, R., Hiramatsu, M., Mori, A., Fujita, Y.,
Yasuhara, A., Yoshida, T., Okuda, T. (1989) Effects of the
interaction of tannins and related polyphenols on superoxide anion
radical, and on 1,1-diphenyl-2-picrylhydrazyl radical. Chem.
Pharm. Bull. (Tokyo) 37: 2016–2021
Hayman, S., Kinoshita, J. H. (1965) Isolation and properties of lens
aldose reductase. J. Biol. Chem. 240: 877–882
Inagaki, K., Miwa, I., Okuda, J. (1982) Affinity purification and
glucose specificity of aldose reductase from bovine lens. Arch.
Biochem. Biophys. 216: 337–344
References
Ahmad, V. U., Shah, M. G., Mohammad, F. V., Ismail, N.,
Noorwala, M. (1991) Macrophylloside. A flavone glucoside from
Primula macrophylla. Phytochemistry 30: 4206–4208
Iwata, S., Nagata, N., Omae, A., Yamaguchi, S., Okata, Y., Shibata,
S., Okuyama, T. (1999) Inhibitory effect of chalcone derivatives on
recombinant human aldose reductase. Biol. Pharm. Bull. 22: 323–
325
Aida, K., Tawata, M., Shindo, H., Tsukahara, S., Onaya, T. (1988)
Effects of anti-inflammatory drugs and ONO-2235 on lens aldose
reductase and on sorbitol accumulation in red blood cells.
Yamanashi Med. 3: 47–56
Jorge, J. P. F., Sbarbati-Nudelman, N. (1986) Cyclization of sub-
stituted 2h-hydroxychalcones to flavanones. Solvent and isotope
effects. J. Chem. Soc. Perkin Trans. 2: 1117–1120
Aida, K., Tawata, M., Shindo, H., Onaya, T., Sasaki, H., Yamaguchi,

668
Soon Sung Lim et al
Larson, L. (1988) The antioxidants of higher plants. Phytochemistry Shin, K. H., Kang, S. S., Seo, E. A., Shin, S. W. (1995) Isolation of
27: 969–978
aldose reductase inhibitors from the flowers of Chrysanthemum
boreale. Arch. Pharm. Res. 18: 65–68
Lee, A. Y., Chung, S. S. (1999) Contributions of polyol pathway to
oxidative stress in diabetic cataract. FASEB J. 13: 23–30
Ou, P., Nourooz-Zadeh, J., Tritschler, H., Wolff, S. (1996) Activation
of aldose reductase in rat lens and metal-ion chelation by aldose
reductase inhibitors and lipoic acid. Free Rad. Res. 25: 337–346
Price, J. R. (1939) The yellow coloring matter of Dahlia variabilis. J.
Chem. Soc. 1017–1018
Singh, M., Singh, J. (1985) Chemical examination of the seeds of
Cassia spectabilis. Z, Naturforsch. B, 40: 550
Spycher, S. E., Tabataba-Vakili, S., O’Donnell, V. B., Palomba, L.,
Azzi, A. (1997) Aldose reductase induction: A novel response to
oxidative stress of smooth muscle cells. FASEB J. 11: 181–188
Srivastata, S. K., Awasthi, S., Wang, L., Bhatnagar, A., Awasthi, Y.
C., Ansari, N. H. (1996) Attenuation of 4-hydroxynonenal-induced
cataractogenesis in rat lens by butylated hydroxytoluene. Curr. Eye
Res. 15: 749–754
Rall, G. J. H., Oberholzer, M. E., Ferreira, D., Roux, D. G. (1976)
Crown ether application in the synthesis of chalcones. Tetrahedron
Lett. 13: 1033–1036
Rastelli, G., Antolini, L., Benvenuti, S., Costantino, L. (2000) Struc-
tural bases for the inhibition of aldose reductase by phenolic
compounds. Bioorganic Med. Chem. 8: 1151–1158
Tawata, M., Ohtaka, M., Hosaka, Y., Onaya, T. (1992) Aldose
reductase mRNA expression and its activity are induced by glucose
in fetal rat aortic smooth muscle (A10) cell. Life Sci. 51: 719–726
Winget, G. D., Izawa, S., Good, N. E. (1969) Inhibition of photo-
phosphorylation by phlorizin and closely related compounds. Bio-
chemistry 8: 2067–2074
Reyes-Chilpa, R., Gomez-Garibay, F., Moreno-Torres, G., Jimenez-
Estrada, M., Quiroz-Vasquez, R. I. (1998) Flavonoids and isoflavo-
noids with antifungal properties from Platymiscium yucatanum
Haertwood. Holzforschung 52: 459–462
Wolff, S. P., Dean, R. T. (1987) Glucose autoxidation and protein
modification. The potential role of ‘autoxidative glycosylation’ in
diabetes. Biochem. J. 245: 243–250
Sato, S., Kador, P. F. (1990) Inhibition of aldehyde reductase by
aldose reductase inhibitors. Biochem. Pharmacol. 40: 1033–1042
Severi, F., Costantino, L., Benvenuti, S., Vampa, G., Mucci, A. (1996)
Synthesis and description of chalcone-like compounds, inhibitors
of aldose reductase. Med. Chem. Res. 6: 128–136
Wollen-weber, E., Seigler, D. S. (1982) Flavonoids from the exudate
of Acacia neoveruicosa. Phytochemistry 21: 1063–1066
Yokozawa, T., Chen, C. P., Dong, E., Tanaka, T., Nonaka, G.,
Nishioka, I. (1998) Study on the inhibitory effect of tannins and
flavonoids against the 1,1-diphenyl-2-picrylhydrazyl radical.
Biochem. Pharmacol. 56: 213–222
Shin, K. H., Kang, S. S., Kim, H. J., Shin, S. W. (1994) Isolation of an
aldose reductase inhibitor from the fruits of Vitex rotundifolia.
Phytomedicine 1: 145–147